Antimicrobial Activity, Cytotoxicity and Phytochemistry of Selected Medicinal Plants in Meru Central District, Kenya

ANTIMICROBIAL ACTIVITY, CYTOTOXICITY AND PHYTOCHEMISTRY OF SELECTED MEDICINAL PLANTS IN MERU CENTRAL DISTRICT, KENYA

Joseph Kisivo Musau, (B.Sc. Nursing -University of Nairobi) r— ■»-/

A thesis submitted in partial fulfillment of requirements for the degree of Master of Science in Pharmacology and Toxicology of University of Nairobi

Department of Public Health, Pharmacology and Toxicology, Faculty of Veterinary Medicine, College of Agriculture and Veterinary Sciences, University of Nairobi

University of NAIROBI Library

May 2011

UNIVERSITY OFNAIflOBlI K.4SETS LIBRARY I 11

DECLARATION This thesis is my original work and has not been presented for a degree in any other university.

Signed J*\ f 3-*-* f (

Joseph Kisivo Musau (BSc-N)

This thesis has been submitted for examination with our approval as university supervisors:

Dr. James M. Mbaria (PhD)

Department of Public Health, Pharmacology and Toxicology

Faculty of Veterinary Medicine,

University of Nairobi

Signed ------¥ ^ W

Dr. Daniel W. Gakuya (PhD)

Department of Clinical Studies,

Faculty of Veterinary Medicine

University of Nairobi Ill

DEDICATION

This thesis is dedicated to my wife Caroline Kirema-Musau and my children, son Edgar Allan

Musau and daughter Sheila Muthoni Musau

On each hank of the stream all kinds of trees will grow to provide food...... The trees will provide food and their leaves will he used for healing people.

(Ezekiel 47:12 Good News Bible) IV

TABLE OF CONTENTS

declaration ...... II

DEDICATION...... Ill

TABLE OF CONTENTS...... IV

LIST OF TABLES...... X

LIST OF FIGURES...... XI

LIST OF PLATES...... XII

APPENDICES...... XIII

ACKNOWLEDGEMENT...... XIV

LIST OF ACRONYMS AND ABBREVIATIONS...... XV

ABSTRACT...... XVI

CHAPTER ONE: INTRODUCTION...... 1

1.0 BACKGROUND...... 1

1.1 RESEARCH PROBLEM AND JUSTIFICATION...... 3

1.2 NULL HYPOTHESIS...... 4

1.3 OBJECTIVES...... 4

1.3.1 Overall objective...... 4

1.3.2 Specific objectives...... 5

CHAPTER TWO: LITERATURE REVIEW...... ,...... 6 V

2 i plants as sources of medicine...... 6

2.2 Plant derived drugs...... 8

2.2.1 Drugs acting on the cardiovascular system...... 8

2.2.2 Drugs acting on the nervous system...... 10

2.2.3 Drugs acting on the gastro intestinal system...... 11

2.2.4 Drugs acting on the urinary and reproductive systems...... 11

2.2.5 Drugs used for treatment of malignancies...... 11

2.2.6 Drugs used on the endocrine system...... 12

2.2.7 Drugs acting on the respiratory system...... 12

2.2.8 Drugs acting on the skin and mucus membranes...... 13

2.2.9 Drugs for treatment of infections...... 13

2.3 Classification of plant derived drugs...... 15

2.3.1 Alphabetical...... 15

2.3.2 Taxonomic...... 15

2.3.3 Morphologic...... 15

2.3.4 Pharmacologic or therapeutic classification...... 16

2.3.5 Chemical or biogenetic...... 16

2.4 Commerce in medicinal plants...... 16

2.5Traditional herbal remedies and conventional modem medicine...... 17

2.6 Screening of herbal products...... 19

2.7 Quality control of herbal medicines...... 20

2.7.1 Organoleptic, macroscopic and microscopic methods ( 22 VI

2.7.2 Ash values...... 22

2.7.3 Herbicide and pesticide residues...... 22

2.7.4 Foaming index and haemolytic activity...... 23

2.7.5 Foreign matter...... 23

2.7.6 Quantification of the active compounds...... 23

2.7.7 Microbial load...... 23

2.7.8 Heavy metals...... 24

2.7.9 Moisture content...... 24

2.8 Regulation of traditional medicine and products...... 25

2.9 Some active principles in medicinal plants...... 26

2.9.1 Alkaloids...... 27

2.9.2 Anthraquinones...... 29

2.9.3 Glycosides...... 29

2.9.4 Tannins...... 30

2.9.5 Triterpenoids...... 30

2.9.6 Saponins...... 31

2.9.7 Flavonoids...... 31

2.9.8 Oils and fats...... 32

2.9.9 Toxalbumins...... 33

2.9.10 Lignans...... 33

2.9.11 Lectins...... 33

2.10 Literature review of the selected plants...... 34 Vll

2.10.1 Ajuga remota Benth...... 34

2.10.2 Harrisonia abyssinica, Oliv...... 35

2.10.3 Erythrina abyssinica, Lam...... 35

2.10.4 Ocimum suave. Wild...... 36

2.10.5 Piliostigma thonningii (Schum.) Milne - redhead...... 37

2.11 Literature review on the selected bacteria species...... 38

2.11.1 Bacillus cereus...... 38

2.11.2 Escherichia coli...... 38

2.11.3 Pseudomonas aeruginosa...... 39

2.11.4 Staphylococcus aureus...... 39

2.12 Antimicrobial assays...... 40

2.12.1 Broth dilution method...... 40

2.12.2 Disc diffusion assay...... 40

2.12.3 Well diffusion assay...... 41

2.13 Ethnopharmacological documentation of meru central district...... 41

CHAPTER THREE: MATERIALS AND METHODS...... 42

3.1 The study area...... 42

3.1.1 Selection and collection of plant materials...... 44

3.2 Preparation of plant extracts...... 47

3.2.1 Extraction...... 47

3.2.1.1 Water extraction 47 Vlll

3.2.1.2 Methanol extraction...... 47

3.3 Antibacterial activity testing...... 48

3.3.1 Bacteria quantification...... 48

3.3.2 Determination of MIC and MBC...... 49

3.4 Cytotoxicity study...... 51

3.4.1 Preparing the marine salt solution...... 51

3.4.2 Hatching the brine shrimp...... 51

3.4.3 Preparing the plant extracts for the test...... 51

3.4.4 Cytotoxicity test...... 52

3.5 Preliminary phytochemical screening...... 53

3.5.1 Test for alkaloids...... 53

3.5.2 Test for sterols and triterpenes...... 53

3.5.3 Test for saponins...... 53

3.5.4 Test for flavonoids and flavones...... 53

3.5.5 Test for tannins...... 54

3.5.6 Test for cardiac glycosides...... 54

3.5.7 Test for anthraquinones...... 7...... 54

CHAPTER FOUR: RESULTS AND DISCUSSION...... 55

4.1 RESULTS...... 55

4.1.1 Ethnobotanical survey...... 55

4.1.2 Antibacterial Testing...... 55

4.1.3 Brine Shrimp Lethality Test...... t...... 61 IX

4.1.4 Preliminary Phytochemical Analysis...... 65

4.1.4.1 Erythrina abyssinica root bark and stem bark...... 65

4.1.4.2 Piliostigma thonningii stem bark and leaves...... 65

4.1.4.3 Ajuga remota leaves...... 65

4 i .4.4 Ocimum suave leaves...... 65

4.1.4.5 Harrisonia abyssinica whole plant...... 66

4.2 DISCUSSION...... 70

4.2.1Ethnobotanical survey...... 70

4.2.2 Antibacterial testing...... 70

4.2.3 Brine shrimp lethality test...... 71

4.2.4 Preliminary Phytochemical analysis...... 73

4.2.4.1 Erythrina abyssinica root bark and stem bark...... 73

4.2.4.2 Piliostigma thonningii stem bark and leaves...... 74

4.2.4.3 Ajuga remota leaves...... 75

4.2.4.4 Ocimum suave leaves...... 75

4.2.4.5 Harissonia abyssinica whole plant...... 76

:HAPTER FIVE: GENERAL CONCLUSIONS...... 78

.EFERENCES 81 X

LIST OF TABLES

Table 2.1: Some of drugs derived from ethnobotanical leads...... 14

Table 2.2: Major alkaloid groups...... 28

Table 3.1: Plants from Meru central district Selected for this study...... 45

Table 4.1: The antibacterial activity of the selected Plants extracts against gram positive bacteria...... 57

Table 4.2: The antibacterial activity of the selected Plants extracts against gram negative bacteria...... 58

Table 4.3: MIC/MBC levels of the aqueous and methanolic extracts of the selected plants

...... 60

Table 4.4: Percentage brine shrimp mortality caused by the methanolic extracts of the

Selected plants...... 62

Table 4.5: Lethal concentration of the selected plant extracts...... 63

Fable 4.6: Phytochemistry results of the selected plants 67 XI

LIST OF FIGURES

Figure 3.1: Map of Kenya showing the location of Meru central district...... 43

Figure 4.1: MIC and MBC of the selected plants’ extracts...... 59

Figure 4.2: Comparison of the percentage brine shrimp mortality due to serial

dilutions of the extracts and vincristine sulphate...... 64 XU

LIST OF PLATES

Plate 3.1: Photographs of the aerial parts of the selected plants...... 46

Plate 4.1: Frothing test for saponins on the selected plants...... 68

Plate 4.2: (a) Positive tannin test for Erythrina abyssinica root bark...... 69

Plate 4.2: (b) Positive Keller-Killian test for Piliostigma thonningii stem bark...... 69 XIII

APPENDICES

Appendix 1: Preparation o f blood agar medium...... 94

Appendix 2: Preparation of Mueller Hinton broth medium...... 94

Appendix 3: Preparation of Tryptone Soya Agar...... 94 XIV

ACKNOWLEDGEMENT

I acknowledge my God for His grace that has enabled me come this far and the University of

Nairobi for giving me an opportunity to study in the field of pharmacology and toxicology. My supervisors, Dr Mbaria and Dr Gakuya were of great assistance and encouragement. Dr Gakuya provided the brine shrimp eggs and the Finney computer program. The financial support I got from the German exchange program (DAAD) in my second year of study enabled this work to come to fruition. 1 am grateful to my wife Caroline, son Allan and daughter Sheila who understood when I was busy. Dr Nguta helped me conceive the idea of research on natural products.

Dr Itonga’s input in this study is invaluable. The staff of the department of public health, pharmacology and toxicology, were of great help during the carrying out of the pharmacological and toxicological studies. I appreciate their support, especially Mr. Nduhiu, Mrs. Mwangi, Mr.

Maloba, Mr. Gitau, Mr. Nderitu and Mr Mainga. I would like to appreciate Mr Musembi of

Department of Land Resource Management and Agricultural Technology (LARMAT) for the botanical identification of the plants.

Mr. Cheboi of the phytochemistry department at National Museums of Kenya guided me in the phytochemical work while Mr. Wango and Vivian at the Department of Animal Physiology,

University of Nairobi, helped in freeze-drying the plant material. My classmates: Dr R Okumu,

Dr D. Mureithi and Dr H. Amenya gave me moral support and encouragement. 1 acknowledge the traditional herbal practitioners in Mwanganthia Location of Meru central district who freely shared their knowledge and information on plants and herbal medicines. God bless you all. XV

LIST OF ACRONYMS AND ABBREVIATIONS a tc c American type culture collection

AU African Union

BA Blood Agar

Cfu Colony forming units

GoK Government of Kenya h iv /a id s Human Immunodeficiency Virus/ Acquired Immunodeficiency Syndrome

ICRAF International Centre for Research in Agro Forestry

MBC Minimum Bactericidal Concentration

MHA Mueller Hinton Agar

MHB Mueller Hinton Broth

MIC Minimum Inhibitory Concentration

THP Traditional Health Practitioner

TMP Traditional Medical Practitioner

TSA Tryptone Soya Agar

WHO World Health Organization XVI

ABSTRACT

In Kenya, over 70% of the population relies on traditional medicine as source of primary healthcare due to its accessibility, affordability and acceptance. Traditional medicine and western medicine are used side by side depending on the cost of conventional medicine. The cost, side effects, toxicity and emerging resistance to antimicrobials has led to natural products being investigated as promising agents for the treatment of diseases.

Meru Central District has inequitable distribution of health facilities and people have to walk at least 7 kilometers to these facilities. High HIV/AIDS prevalence and low doctor / patient ratio means many people seek medical services from TMP. This study aimed at identifying and screening medicinal plants from this area for their pharmacological, toxicological and phytochemical properties.

Twenty three (23) herbal practitioners identified 86 plants from 37 plant families of ethnomedical interest. The plants were botanically identified by a plant taxonomist at the

Department of Land Resource Management and Agricultural Technology (L ARM AT),

University of Nairobi. Selection of plants for this study was based on the informant consensus among the identified herbal medicine practitioners on their use for treating microbial infections in human.

Five (5) plants and seven (7) plant parts were selected and evaluated for their antimicrobial activity, cytotoxicity and phytochemical properties. They are Piliostigma thonningii (leaves and stem bark), Ajuga remota (leaves), Ocimum suave (leaves), Erythrina abyssinica (root bark and stem bark) and Harissonia abyssinica (whole plant). XVII

Standard bacteria cultures of Staphylococcus aureus, Bacillus cereus, Escherichia coli and

Pseudomonas aeruginosa were used to determine the minimum bactericidal concentration

(MBC) and minimum inhibitory concentration (MIC) of the plants’ methanolic and water extracts through standard microbial techniques at the Department of Public Health,

Pharmacology and Toxicology University of Nairobi. Comparison was computed for susceptibility of bacterial species and considered significant at p<0.05.

Cytotoxicity of the methanolic extracts was determined using brine shrimp lethality test with serial dilutions of lOOOpg/ml, lOOpg/ml and lOpg/ml. Lethal concentration 50 (LC50) at 95% confidence intervals was determined using Probit analysis. Established phytochemical tests were performed to show the presence or absence of secondary metabolites.

All the plants showed activity against the test bacteria except Erythrina abyssinica stem bark.

Piliostigma thonningii stem bark was most active against the test bacteria. There was no difference in antibacterial activity between the methanolic and water extracts. Gram positive bacteria were more susceptible to the extracts than gram negative bacteria. Bacillus cereus was most susceptible while Escherichia coli most resistant. All the extracts had significant bioactivity at LC5o<1000pg/ml. Ajuga remota was most toxic with LC50 of 69.24pg/ml. Tannins and saponins were present in all extracts. Flavones and anthraquinones were absent.

The plants had appreciable antibacterial activity. The results of this study support the use of these plants by the herbalists. Piliostigma thonningii stem bark exhibited the most antibacterial activity. Further studies should be carried out on this plant regarding its antimicrobial potential.

More research is needed to document and scientifically validate the plants used by the Meru community. 1

CHAPTER ONE: INTRODUCTION

1.0 Background Plants have a long history of use in treatment and management of different disease conditions.

They have been used for treatment of many diseases all over the world since ancient times and about 25% of current drugs are derived from plants (Wanyoike et al., 2004). In certain African countries up to 90% of the population relies exclusively on plants as sources of medicines

(Hostetmann et al., 2000). This ethnobotanical utilization of plants for treatment of various diseases, antidote against magic and for religious ceremonies forms the basis of traditional medicine (Kokwaro, 1993).

Traditional medicine is the sum total of knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures that are used to maintain health as well as to prevent, diagnose, improve or treat physical and mental health (WHO, 2009). It is the aggregate of beliefs, practices, measures, activities, pursuits and procedures of all kind which from prehistoric times enabled man to alleviate suffering prevent and cure diseases. Traditional healers have for centuries been the main providers of primary health care in Africa (Kala et al.,

2004).

Traditional medicine is an integral part of every culture in Kenya. It is affordable, socially acceptable and easily accessible. It is efficacious and holistic in its approach and caters for among other things the spiritual, emotional and physical care of the clients (Kareru et al., 2007;

Miaron et al., 2004; Munguti, 1997; Njoroge and Busman, 2007). Like any other community in

Kenya, the Meru community has a large traditional medicine base. A survey in Meru Central

District identified 86 plants in 37 families, as having ethnomedical and ethnoveterinary uses

(Itonga, personal communication). / 2

plants contain pharmacologically active compounds. Knowledge of the chemical constituents of plants is desirable for the discovery of therapeutic agents and other substances of economic value such as tannins, gums and oils (Chetri et al., 2008). Identifying the chemical constituents of plants and understanding the probable actions of such constituents on humans and animals provides the rationale for traditional use of such plants in treating and managing disease conditions (Kisangau et al., 2007; Njoroge and Busman, 2007).

The appearance of multidrug resistant pathogens and others with reduced susceptibility to available antimicrobials adds to the urgency for the search of new infection fighting strategies.

Due to the increase in HIV/AIDS prevalence and re-emergence of diseases like tuberculosis, the spectra of untreatable or difficult to treat microbial infections is increasing. The side effects and toxicity of available antimicrobials and their high cost further complicates the current methods of fighting infections (Bii, 2001; Janovska et al., 2003; Runyoro et al., 2006).

Efforts are now directed towards finding new and innovative antimicrobial agents with novel mechanisms of actions. Natural products of higher plants are probable sources of antimicrobial agents which have added advantages of being safe and biodegradable (Adenisa et al., 2000).

Plants may serve as natural blue prints in the development of new drugs or as phytomedicines to be used to treat disease (Abubakar et al., 2008). Infections associated with bacterial pathogens are among some of the indications for treatment with traditional remedies that include plant products (Njoroge and Busman, 2007).

Many drugs owe their origin to plants and Wanyoike et al (2004), estimated that about 25% of drugs are derived from plants. In some cases, natural materials continue to be the only viable 3

commercial source of the active compound. Glaxo Wellcome harvests 10000 metric tons dry

weight of poppy capsule per year to provide a source of opiate alkaloids and about 170 metric tons per year of Digitalis lantana leaf as the source of digoxin (Evans, 2005).

Chemical studies of medicinal plants provide a valuable base for the discovery and development

of new drugs of natural origin. This is because the medicinal value of plants lies in the chemical

substances that produce a definite action on the body (Musyimi et al., 2008). The use of

medicinal plants by herbalists must be validated, regulated and evaluated to avoid the

administration of dangerous concoctions and to provide scientific proof of their usage. Such

validation includes antimicrobial assays, antiprotozal activity, anthelminthic activity and

cytotoxicity (Alluri et al., 2005; Camacho et al., 2003; Moses et al., 2006; Wasswa and Olila,

2006).

1.1 Research problem and justification

The substantial contribution to human health and well being made by medicinal plant species is

now widely appreciated and understood. The demand for medicinal plant species is increasing

and more people are interested in use of medicinal plants to treat and manage diseases. There is

increase in knowledge among the general population regarding the side effects and toxicity of

conventional drugs and the perceived safety of natural products.

More than 70% of Kenyan population relies on traditional medicine as the primary source of

healthcare and 90% of Kenyan people use medicinal plants at one time or another. Plant cures

and other traditional herbal remedies are particularly suitable for use in rural communities

because they are inexpensive, sustainable and culturally appropriate alternatives to conventional

treatments which are expensive. Meru central district has a doctor/ patient ratio of 1:33,259 and 4

IIIV/AIDS prevalence of 38%. The health facilities in the district are not equitably distributed and the road network is poor.

The average distance to the nearest health facility is 7 km and most of the people have to walk to these facilities (GOK, 2005; 2007). This low doctor/patient ratio, the high HIV/AIDS prevalence, the inequitable distribution of health facilities and the inaccessibility to basic health services to the populace forces many people in the district to seek medical attention from traditional medical practitioners.

Antimicrobial resistance has become a global problem. Plants and other natural products are now being thoroughly researched as possible sources of new biologically active compounds with novel mechanisms of action. The major starting point for this research is folklore medicine including traditional remedies. Many traditional remedies in Kenya have not been scientifically validated. Elaborative ethnobotanical research has not been carried out in Meru Central District and there is little documentation on antimicrobial, cytotoxicity and phytochemical properties of medicinal plants used by the community.

1.2 Null hypothesis It was hypothesized that the selected plants had antimicrobial activity, were cytotoxic and contained important phytochemical groups. ^

1.3 Objectives

1.3.1 Overall objective The overall objective was to study the Pharmacological, Toxicological and Phytochemical properties of medicinal plants in Meru Central District, Kenya. 5

1.3.2 Specific objectives 1 To analyze the antimicrobial activity of the selected plants.

2. To carry out the cytotoxicity study of the plants extracts.

3. To carry out a preliminary phytochemical analysis of the selected plants. 6

CHAPTER TWO: LITERATURE REVIEW

2.1 Plants as sources of medicine

Plant molecules, their semi-synthetic and synthetic derivatives are important sources of drugs.

They form part of herbal medicines. According to WHO ( 2009), Herbal medicines include herbs, herbal materials, herbal preparations and finished herbal products that contain parts of plants or other plant materials as active ingredients. From time immemorial, plants have been used as sources of preventive and curative medicinal preparations (Dery et al., 1999). They have been used for both healing and killing depending on the knowledge of their dosage and synergistic action (Barquar, 1995).

Presently, plants continue to be used where in certain developing countries up to 90% of the population relies exclusively on plants as sources of medicine (Hostettmann et al., 2000., Odera

1997). The use of medicinal plants is not unique to the developing countries. It is estimated that

33% of drugs produced in developed countries are from higher plants (Mwangi, 2004). Over

50% of the drugs in clinical trials for cancer were isolated from natural sources, and include

Vinblastine, Vincristine, Etoposide, Teniposide and Taxanes (De Mesquita et al., 2009).

Plants have been used as medicine all over the world since before recorded history (Chetri et al.,

2008). Years of experimentation with plants enabled man to know useful and harmful plants as well as their healing properties. Plant use is universal and all major systems of medicine in the world include aspects of plants. Several well known species, including Licorice (Glycyrrhiza glabra), Myrrh (Commiphora species), and Poppy capsule latex (Papaver somniferum), were referred to by the first known written record on clay tablets from Mesopotamia in 2600BC. 7

These plants are still used today for various diseases and as ingredients of official drugs or herbal preparations used in systems of traditional medicine (Young-Won et al., 2006).

References to the use of medicinal plants as cure for diseases is found in the manuscript of "Eber

Papyrus" of about 16,h century B.C, which recorded the use of Papaver somniferum, Ricinus communis, Urginea sp .and Aloe sp. Theophrastus (370-287 BC) described over 500 plants. Piny the elder (23 - 79 AD) in his book Histroria Naturalis, described 1000 plants with their medicinal, anatomical and horticultural properties. Dioscorides (1st century AD) described 600 plant species in the book De Material Medica. Systematic pharmacopoeias have been developed as early as 3000 BC by the Chinese. Chinese medicinal plant texts date from 4lh century BC.

In India, the earliest description of curative properties of plants is in Rig-Veda (2500 - 1800BC).

The first London Pharmacopoeia appeared in 1618 and the first British Pharmacopoeia in 1864

(Barquar, 1995; Evans, 2005; Kisangau 1999; Shukla et al., 2000). This long history of medicinal plants is known to have resulted in the development of formal systems of medicine particularly in Egypt, Arabia, India, China and Europe. Adams et al, (2009) in a survey of historical usage of herbal medicine in Europe between 16th and 17th century were able to identity 63 plants that had clear indications.

Use of plants as sources of medicine is a major African socio-cultural heritage where ifhas been the mainstay of healthcare system for centuries. This knowledge of plants as medicine was passed orally from one generation to the next and often within a family (Elujoba et al., 2005;

Miaron et al 2004; Munguti, 1997). Despite the long history of plant usage in treating and preventing illnesses, less than 5 % of the world’s plants species have been examined for their activity and constituents (Goodwin and Mercer, 2003). 8

2.2 Plant derived drugs plants are major sources of drugs with various functions in the human body. Scrutiny of medical indications by source of compounds has demonstrated that natural products and related drugs are used to treat 87% of all categorized human diseases, including as antibacterial, anticancer, anticoagulant, antiparasitic, and immunosuppressant agents, among others (Young-Won et al .,

2006).

2.2.1 Drugs acting on the cardiovascular system Diseases of the cardiovascular system are a major cause of mortality especially in the developed countries. This has led to intense interest in research on treatment and prevention of cardiovascular diseases. In addition there is increased awareness in the population concerning healthy foods and healthy living, exercise and medicinal plant products to complement the conventional drugs in management and prevention of these diseases.

The plant kingdom is being researched on actively with the aim of identifying compounds that may serve as lead compounds for the synthesis of new drugs. At present various products have been identified that have several activities on the cardiovascular system. Quinidine is derived from the cinchona species and is an example of antiarrhythmic drugs derived from plants. It acts on both supraventricular and ventricular arrhythmias. It is currently used in atrial fibrillation

(Pengelly, 2004).

Cardiac glycosides increase the force and contractility of the heart. They do this by inhibiting the membrane bound Na+K - ATPase to cause increase in intracellular Ca+ hence increased cardiac muscle contraction. Cardiac glycosides increase atrial and ventricular myocardial excitability and also decrease the rate of atrial ventricular contraction. They increase vagal tone and myocardial sensitivity to vagal impulses. Among the cardiac glycosides, the most commonly used are those 9 from Digitalis species. They are Digitoxin from Digitalis purpurea and Digoxin from Digitalis lantana both of Schrophulariaceae family.

Other cardioactive glycosides are Stropanthin from Strophanthus gratus of Apocynaceae family,

Convallotoxin and Convaloside from Convallaria majalis of Liliaceae family; Odoroside and

Oleandrin from Nerium oleander of Apocynaceae Family. Some plant derived drugs have antihypertensive activity. Reserpine from the roots of Rauwolfia serpentina is used as a hypotensive agent. Other plants with hypotensive activity are Crataegus, Tilia, Fagopyrum and veratrum species (Evans, 2005).

Drugs acting on the cellular elements of blood are the platelet activating factor antagonists and the oral anticoagulants. Platelet aggregation leads to adhesion of platelets on the walls of blood vessels and formation of thrombi. This causes narrowing of the blood vessels and when thrombi are dislodged from its attachment in to circulation, heart malfunction occurs. Platelet aggregation is triggered by platelet activating factor which is released from activated basophils. Aspirin is frequently used to prevent thrombi formation. Various plants have been found to have activity against platelet activating factor. Species of Forsythia, Arctium, Centtipeda, Tussilago, Pyrola,

Populous and Peucedanum and the neolignan kadsurenone from Piper futokadsura have anti platelet activating factor activity (Evans, 2005; Pengelly, 2004).

Oral anticoagulants inhibit the clotting mechanism and don’t have activity on platelet aggregation. They are of great value in prevention and management of arterial thrombosis.

Coumarin derivatives antagonize the effects of Vitamin K therefore preventing coagulation.

Plant derivatives of Melilotus officinalis, Gallium aparine and Lavandula officinalis are used in herbal medicine and have activity against Vitamin K. (Evans, 2005) 10

2.2.2 Drugs acting on the nervous system Drugs acting on the central nervous system are classified as stimulants or depressants. Further classification is based on specific action such as anticonvulsants and other psychopharmacological actions. Drugs acting on the central nervous system include narcotic analgesics such as Morphine and Codeine from Papaver somniferunr, analeptics like Strychnine from Slrychnos nox-vomica and Lobeline from Lobelia inflata and depressants of motor activity like Atropine and Hyoscine from Atropa belladonna (Evans, 2005).

The transmitters of autonomic nervous system are acetylcholine and noradrenaline together with its derivatives. The plant derived drugs acting on the autonomic nervous system either mimic the action of the neurotransmitters or antagonize them. Acetylcholine-like drugs are Pilocarpine from Pilocarpus jaborandi, Arecoline from Areca catechu and Muscarine from Amanita muscaria. Antagonists of acetylcholine include tropane ester alkaloids such as Atropine and

Hyoscine from Atropa belladonna and neuromuscular blocking agents like Tubocurarine which is obtained from the bark of Chondodendron tomentosum. Tubocurarine is a long duration non- depolorarizing neuromuscular blocking agent which is a competitive antagonist of nicotinic neuromuscular acetylcholine receptors (Evans, 2005; Pengelly, 2004).

Adrenaline like drugs includes Ephedrine from aerial parts of Ephedra species and antagonists of adrenaline are ergot alkaloids such as ergotamine. Ergot alkaloids are derived from the dried sclerotium of fungi Claviceps purpurea when it grows in the seeds of rye plant and other grass plants Reserpine from the roots of Rauwolfia serpentina depletes noradrenaline (Evans, 2005;

Pengelly, 2004) 11

2.2.3 Drugs acting on the gastro intestinal system The bitters stimulate gustatory nerves in the mouth to give rise in the psychic secretion of gastric juice. Examples are the bitters from extracts of Gentian, Quassia, Cinchona and Nox vomica.

Anticholinergics like Hyoscine and Hyoscyamine from Atropa belladonna regulate gastric motility and spasms. Ipecacuanha preparations are used as emetics while ginger is useful in prevention of motion sickness. Dill oil derived from Anethum graveolens is used to prevent flatulence in young babies.

Extracts from Senna species and castor oil from Ricinus communis are examples of laxatives and purgatives derived from plants. Arachis oil from the seeds of Arachis hypogoea and Esculine from the bark of Aesculus californica are used together with other drugs in suppositories.

Morphine and codeine from Papaver somniferum reduce gastric motility in addition to their analgesic activity (Evans, 2005; Pengelly, 2004)

2.2.4 Drugs acting on the urinary and reproductive systems Ergometrine which is derived from Claviceps purpurea is used as a uterine stimulant in child birth. Juniper is derived from Juniperus species and is a urinary antiseptic. It is also used in treatment of cystitis and urethritis. Yohimbine is derived from Aspidosperma querbracho is used in management of erectile dysfunction. Prunus Africana bark, Serenoa repens fruits and Urtica dioica roots are used for management of benign prostate hypertrophy (Evans, 2005; •Pengelly,

2004).

2.2.5 Drugs used for treatment of malignancies Plants have been used for the treatment of malignancies for centuries. Podophylum was used

over 2000 years ago by the Chinese as an antitumor drug. Etoposide and Teniposide are semi

synthetic derivatives of Podophyllotoxin which is derived from Podophyllum hexadrum and 12

Podophyllum emodi. Etoposide is used for small cell lung cancer and testicular cancer.

Teniposide is used for pediatric cancers.

The alkaloids of Cartharanthus roseus yield Vinblastine and Vincristine which are currently used alone or in combination for treatment of various cancers such as Hodgkins Disease and child hood leukemia. Taxol from Taxus brevifolia is approved for treatment of breast cancer.

Camptothecin derivatives like Topotecan and Irinotecan are used for ovarian cancer and colorectal cancer respectively. Camptothecin is from Camptotheca acuminate (Cragg and

Newmann, 2006; Evans, 2005; Pengelly, 2004).

2.2.6 Drugs used on the endocrine system Metformin is approved for management of non insulin dependent diabetes mellitus. It is derived from Galega officinalis that was historically used for treatment of diabetes in medieval Europe

(Shukla et al., 2000).

2.2.7 Drugs acting on the respiratory system Ephedra and Ephedrine from Ephedra species and Xanthenes such as Theophilline from

Camellia sinensis are used as nasal decongestants and bronchodilators. Codeine from Papaver somniferum is used in cough mixtures as antitussive. Menthol from Mentha spp and eucalyptus oil from Eucalyptus spp are used in inhalations and cough preparations for their soothing action

(Pengelly, 2004). Tiotropium, a derivative of atropine from Atropa belladonna (Solanaceae) and related tropane alkaloids from other solanaceous plants, is a potent reversible nonselective inhibitor of muscarinic receptors.

Tiotropium bromide has been approved by the United States Food and Drug Administration

(FDA) for the treatment of bronchospasm associated with chronic obstructive pulmonary disease 13

(COPD). Tiotropium is structurally analogous to ipratropium, a commonly prescribed drug for

COPD, and has shown longer lasting effects (Evans, 2005; Pengelly, 2004; Young-Won, 2006)

2.2.8 Drugs acting on the skin and mucus membranes Drugs affecting the skin may be emollients, absorbents, astringents, irritants or antiseptics.

Emollients and demulcents include fixed oils like Arachis oil fats. Waxes, gums such as acacia from Acacia species, and mucilage like psylium from Plantago ovata are used as emollients and demulcents. They are the vehicles used in preparation of ointments, creams and lotions.

Absorbents are mainly starch, alginates from sea weed, and charcoal. Astringents are mainly tannins. Antiseptics of plant origin are eucalyptus oil from Eucalyptus spp, thyme oil from

Thymus spp and tars (Evans, 2005; Pengelly, 2004).

2.2.9 Drugs for treatment of infections Quinine which is isolated from Cinchona spp has been one of the most effective agents for management of malaria. It is still used in some third world countries to combat malaria which is resistant to other drugs. Artemisinin and its derivatives from Artemisia annua (Asteraceae) are the drugs of choice for treatment of malaria. It is effective for treatment of chloroquine resistant malarial strains of Plasmodium vivax and Plasmodium falciparum.

Emetine which is an alkaloid from the root of Cephaelis ipecacuanha (Rubiaceae) is used as its hydrochloride or bismuthiodide for treatment of amoebic dysentery and Berberine from Berheris vulgaris is indicated for treatment of bacillary dysentery. Echinacea spp are used as adjuvant therapy in treatment and prophylaxis of upper respiratory tract infections. Thymol from Thymus vulgaris is used as a topical antifungal agent while Agrimophol from Agrimonia supatoria is an anthelminthic (Evans, 2005; Pengelly, 2004). Some of the drugs derived from plants and their medical uses are summarized in table 2.1. 14

Table 2.1: Some of the drugs that have been developed from ethnobotanical leads

DRUG PLANT SOURCE MEDICAL USE

Aspirin Filipendula umaria Analgesic, anti-inflammatory, antipyretic.

Atropine Atropa belladonna Premedication in surgery, pupil dilator

Caffeine Camellia sinensis CNS Stimulant

Codeine Papaver somniferum Analgesic, antitussive

Colchicine Colchicium autumnale Gout management

Digoxin Digitalis lantana Atrial fibrillation

Ephedrine Ephedra sinica Bronchodilator

Ipecac Psychotria ipecacuanha Emetic

Physostigmine Physosiigma venenosum Glaucoma

Pilocarpine Pilocarpus jaborandii Glaucoma

Psoralen Psoralea corylifolia Vitiligo

Quinine Cinchona pubescens Malaria

Reserpine Rauwolfia serpentina Antihypertensive

Scopolamine Datura stramonium motion sickness

Theophilline Camellia sinensis Management of asthma

Vinblastine Catharanthus roseus Hodgkin’s disease

Vincristine Catharanthus roseus Leukemia

Sources: Evans. 2005; Kisangau, 1999; Pengelly, 2004; Sharma, 2007, 15

2.3 Classification of plant derived drugs

Classification of drugs derived from plants is by alphabetical, taxonomic, morphologic, pharmacologic/therapeutic and chemical/biogenetic (Evans, 2005)

2.3.1 Alphabetical This is either Latin or vernacular names. It is used in dictionaries and pharmacopoeias. It is suitable for quick reference. Pharmacopoeias contain information on the composition, preparation and storage of pharmaceuticals. Pharmacopoeias were first formulated by autonomous cities and were intended to provide regulation on the various forms of medicinal preparations available at that time.

However many of pharmacopoeias in existence today do not include herbal preparations except few like the British pharmacopoeia and the Chinese pharmacopoeia. Many countries in Africa do not have national pharmacopoeias or national drug monographs. Mozambique has a national pharmacopoeia that is legally binding (Evans, 2005; Wagate, 2008). Few have research institutions dealing with herbal medicines. Kenya has not yet developed a national pharmacopoeia and herbal medicine is unregulated. However efforts are been made to regulate and control trade and profession of herbal medicine through an Act of Parliament.

2.3.2 Taxonomic This is classification on basis of an accepted botanical system. The drugs are arranged according to the plants from which they are obtained. They are also arranged in classes, orders, families, genera and species of the plants (Evans, 2005; Wagate, 2008).

2.3.3 Morphologic The drugs are divided into groups such depending on the part of plant. The most commonly used plant parts are leaf (folia), flower (flos), fruit (fructus), herb (herba), bark (cortex), rhizome 16

(rhizome) roots (radix), and bulb (bulbus). Others are latex, resins, gums, woods, seeds, oils, waxes and extracts (Evans, 2005; Wagate, 2008).

2 3.4 Pharmacologic or therapeutic classification This is classification according to the action of the most important constituent or their therapeutic use. This type of classification is most common as more plants are being screened for their specific pharmacologic activity. (Evans, 2005)

2.3.5 Chemical or biogenetic This is classification according to the important constituents such as alkaloids, glycosides, volatile oils or their biosynthetic pathways. This classification is biased towards phytochemistry

(Evans, 2005).

2.4 Commerce in medicinal plants Trade in medicinal plants is a major business. It is estimated to be worth US$60 billion (WHO,

2004). This is because some of the constituents of these plants cannot be synthesized and therefore are only available in natural form. Cameroon is the major source of the bark of Primus africana in the world market. The market value in Europe and America for Prunus africana is

150 million US dollars per year. Kenya exports 1923 tons of Prunus africana per year while

Uganda and Madagascar 193 and 800 tons respectively (Evans, 2005). The bark is used for treating prostate gland hypertrophy (Evans, 2005; Hostetmann et al„ 2000).

Harpagophytum procumbens is used as a tonic, as a treatment for arthritis and rheumatism, to reduce fever, ease sore muscles, reduce cholesterol, and externally the ointment is used to treat sores, boils, and ulcers. It is also used to cleanse the lymph system and to remove toxins from the blood. It is produced in southern Africa, and Namibia is the biggest exporter in the region 17

(Hostetmann et al., 2000; WHO, 2004). Between 10,000 and 15,000 harvesters rely on sales from its collection as their only source of cash (WHO, 2004).

The global demand for Digitalis lantana leaf is about 10000 tonnes per year. An analysis of the top selling pharmaceuticals in 1999 shows that 13 of the top selling 30 medicines with combined sales of over $26 billion have active ingredients whose chemical structure is based on a compound found originally in nature (Evans, 2005).

2.5 Traditional herbal remedies and conventional modern medicine Herbal remedies are well known natural sources for the treatment of various diseases since antiquity all over the world, and African traditional medicine is among the oldest medicinal system (Gurib-Fakim, 2006; Maregesi et al., 2008; Wanyoike et al., 2004). Many people use herbal remedies and it is estimated that over 80% of the people all over the world largely depend on traditional herbal medicine to meet their primary health care needs (Hamayun et al., 2006).

Traditional medicine has remained as the most affordable and easily accessible source of treatment in the primary healthcare system of poor communities. It is the only means of medical treatment for such communities (Yinegar and Yewhalaw, 2007).

Herbal remedies play a major role in traditional medicine in rural areas of Kenya where they are often the therapeutic treatment of choice. Despite the large public and private health expenditures, the health services are still not close to people and healthcare has remained inaccessible to the majority of the population. The health facilities suffer from shortage of trained personnel and inadequate supplies of essential medicines. Besides, some services are not culturally and socially acceptable to some communities (Sindiga, 1995). 18

In the rural communities, traditional medicine offers a holistic approach to healthcare is socially acceptable, accessible, affordable and presumed to be efficacious. This is in contrast to conventional medicine which lacks all the above except efficacy. Traditional medicine has the widest spatial coverage compared to conventional medicine because each community has its own healers (Mutai et al., 2009; Sindiga, 1995).

However traditional medicine does not keep with scientific and technological advancements.

This means that some of the methods and techniques that previously worked have become obsolete. Its rationale is not well defined and some of its aspects like dosage including combinations are not known. Besides, there is no continuity of care since traditional medicine does not keep written records (Sindiga, 1995). It is often passed from generation to generation and often within a family (Miaron et al., 2004). It therefore becomes a secret among the practitioners. This is the greatest impediment to the advancement of traditional medicine.

Moreover, the subject of traditional medicine is not taught in health training institutions here in

Kenya (Mwangi, 2004).

Traditional herbal medicine is a confluence of two factors: the belief in the efficaciousness of herbal medicine and the high cost and inaccessibility of healthcare (Munguti, 1997). In the developing countries, traditional herbal medicine is often used side by side with western medicine and it takes the upper hand when the cost of western medicine is beyond reach (Busia,

2005).

Plants contain many compounds. One of the most promising areas in the search for new

biologically active compounds is the plants used in traditional medicine (Alonso, et al., 1995).

Antimicrobials of plant origin are not associated with many side effects and have an enormous 19 therapeutic potential to heal many infectious diseases since they are presumed to be safe. Even if they were toxic, they will not have great environmental impact since they are biodegradable (Iwu etal, 1999).

For plants to be used alongside modem medicine, careful, phytochemical, pharmacological and toxicological standardization for the chosen plants must be instituted so that dosage can be described in an informed way (Midiwo et al., 2005). The use of modem isolation techniques and pharmacological testing means that new plant drugs usually find their way into medicine as purified substances rather than galenical preparations (Evans, 2005).

2.6 Screening of herbal products

Medicinal plants have been the subject of human curiosity and need for a long time (Omino and

Kokwaro, 1991). Every community in Kenya has a large traditional base of medicinal plants.

Medicinal plants contain active principles which can be used for common infections. However, their use must be validated, regulated and evaluated to avoid the administration of dangerous concoctions by the herbalists (Kareru et al., 2008, b). Despite the well documented ethnobotanical literature, very little scientific information such as efficacy or phytochemistry has become available on indigenous medicinally used plants (Midiwo et al., 2005; Van Vuuren,

2008).

There is a need to systematically evaluate plants used in traditional medicine. Such research could lead to new drug discovery or advance the use of indigenous herbal medicines for orthodox treatments (Parekh and Chanda, 2006). It is necessary to carry out toxicity studies on medicinal plants, even where they have been used for decades, to determine acceptable and non- acceptable toxicity levels (Ogwal-Okeng et al., 2003). Such validation and testing can be 20 achieved by various methods like chemical standardization, biological assays animal models and clinical trials (Kareru et al., 2008, b).

Brine shrimp lethality test is a general bioassay that is capable of detecting a broad spectrum of bioactivity present in crude extracts of plants. It is efficient, rapid and inexpensive and requires small amounts of samples. This test has been used in bioassay fractionation of active cytotoxic and antitumour agents such as Trilobacin from the bark of Asimia triloba, Annona muricata and ent-kaur-16-en-19-oic acid from Elaeoselinum foetidum (Pisutthanan et al., 2004). Antimicrobial assay of plants can offer a good source of new, safe and biodegradable antimicrobial drugs

(Hamza et al., 2006). Phytochemical determination of plant extracts forms basis of action of such extracts. Investigation of secondary plant metabolites has led to important breakthroughs in pharmacology (Farombi, 2003; Kisangau, 1999; Kokwaro, 1993; Nwaogu et al., 2007; Runyoro et al., 2006).

2.7 Quality control of herbal medicines Currently, 33% of drugs produced in developed countries are derived from compounds that are originally isolated from plants. Twenty five percent (25%) of these owe their origin from tropical rain forests of Africa, Asia and South America. Their estimated value is about US$65 billion

(Mwangi et al., 2005). People are getting more informed on the advantages of herbal medicines and supplements as natural sources for good nutrition and management of diseases. Many people irrespective of their social status are turning to herbal and complementary medicines because they perceive them as having few side effects and less toxic in comparison to conventional medicines (WHO, 2002).

The complementary medicine practitioner to patient ratio is better than doctor patient ratio meaning that there is better access to traditional/ complementary medicine. In the developed 21 countries like USA, Germany, France and Belgium, professional training on herbal medicines is offered. In these countries, up to 80% of prescriptions are phytomedicines and about 70% of the populations are regular users of complementary and alternative medicines. Herbal medicine is very advanced in countries like China where there are hospitals, universities and pharmaceutical companies dealing in herbal medicines (Mwangi et al., 2005).

In Kenya, it is common to see hawking of herbal preparations in markets, buses and other public places. As more and more people use herbal medicines, there is a need for quality control especially in developing countries such as Kenya. Among issues that need to be addressed in determination of quality of herbal medicine is hygiene, during and after preparation of the herbal product; the presence of chemical residues such as fertilizers, pesticides and herbicides and the expected limits of heavy metals in the herbal preparations.

Assessment is needed for the level of contamination by bacteria or fungi found in the locality of growth of the plants and presence or absence of foreign matter on the herbal material. Justin-

Temu et al, (2009) identified dust, unspecified additives, and untreated water as possible sources of contamination of herbal medicines in Tanzania. Besides, it was not possible to determine the shelf life of these herbal preparations.

Quality control should also be carried out to ascertain the chemical constituents of "the plant material. Plants contain chemical groups such as glycosides, alkaloids, sulphur groups, saponins, among others which occur in varying amounts. Their concentrations also vary on locality of the

Plant. When in high amounts, they may cause side effects and toxicity. Since the practice of herbal medicine is passed orally from generation to generation and often within a family, it is 22 important to begin the process of quality control with folklore concerning the medicinal plants.

Various tests can be done to assure quality o f the herbal preparation. Some o f the methods are:

2.7.1 Organoleptic, macroscopic and microscopic methods These entail the use of sight, smell, taste and touch to assess the general characteristics of the herbal material. The shape, size, colour, surface, texture and characteristics of the cut surface are compared with an authentic sample. Visual evaluation of the plant is done to ensure that the plant is of the required species and that the right part is being used. Microscopic analysis through examination of microscopic structures like leaf stomata is done to identify the correct part, especially when different parts of plant have different indications (Evans, 2005; Kibwage et al.,

2005).

2.7.2 Ash values The plant material is burnt and the residual ash is measured as total and insoluble ash. These are done to check the presence of undue extraneous matter such as soil, sand, metals and oxalates.

Total ash value is important in substances which have little or no calcium oxalate such as ginger.

Water soluble ash is also specified for ginger. Acid insoluble ash is obtained after boiling the total ash with hydrochloric acid and burning the remaining insoluble matter. It is useful in detecting excess soil and silica in plant material. Sulfated ash measures the extent of contamination by non-volatile inorganic material (Bandaranayake, 2006; Evans, 2005;-Kibwage et al, 2005).

2.7.3 Herbicide and pesticide residues The most common are organochlorines, organophosphates, carbamates and pesticides of plant ongin such as pyrethrum. They are due to agricultural practices like spraying, treatment of soils

^ d fumigation during storage. These can be determined using appropriate methods such as gas

liquid chromatography, gas chromatography, gas chromatography coupled with mass spectrometry so that they do not exceed the expected limits (Evans, 2005; Kibwage et al., 2005).

2.7.4 Foaming index and haemolytic activity These two tests are important for plants that contain saponins. They are important to determine

for aqueous decoctions because it is difficult to prepare and dose accurately in presence of

foaming (Bandaranayake, 2006; Kibwage et al., 2005).

2.7.5 Foreign matter Material that is extraneous to the medicinal plant such as other plant parts, organisms, and

mineral admixtures such as sand, stones and dust must be determined and removed. Herbal

material should be free from moulds, insects and any other visible contaminant. Other ways to

detect foreign matter include macroscopic and microscopic examinations and thin layer

chromatography especially when the foreign matter is a chemical (Kibwage et al., 2005).

2.7.6 Quantification of the active compounds This is done by methods such as thin layer chromatography, liquid chromatography and gas

chromatography. High performance liquid chromatography is essential for quantitative analysis

of complex mixtures. High performance thin layer chromatography is useful to determine

homogeneity of plant extract (Kibwage et al., 2005).

2.7.7 Microbial load This is done to determine presence of pathogens in the herbal material. Such microbial

contaminants include fungi, bacteria and viruses. The source of microbial contamination is poor

methods of harvesting, cleaning, drying, handling and storage of the herbal material. The total

microbial count and total fungal count should be done especially because materials of vegetable

°ngin tend to contain higher levels of microbial contamination than synthetic ones. 24

Fungal toxins like aflatoxin and other microbial toxins like bacterial endotoxins and mycotoxins gj.q of important health concern. Their presence should be determined and thin layer chromatography employed for confirmation (Evans, 2005; Kibwage et al., 2005).

2.7.8 Heavy metals Contamination with heavy metals is either accidental or deliberate. High levels of lead are found in material collected by the roadside or downwind of factories using fossil fuels. Environmental pollution is the major cause of contamination of herbal products with heavy metals like lead, cadmium, copper, mercury and arsenic. Limit tests should be estimated against set standards and requirements. Atomic absorption spectrometry, inductively coupled plasma and neuron activation analysis are also used when the heavy metals are present in trace amounts

(Bandaranayake, 2006; Evans, 2005; Kibwage et al., 2005).

2.7.9 Moisture content Moisture in combination with suitable temperature will lead to proliferation of microorganisms.

Various methods for determining include loss on drying which may be done by spreading the material on glass plates and placing the plates on desiccators over phosphorus hydroxide.

Vacuum drying over an absorbent material may also be used. Another way is to pass a dry inert gas through the heated sample to collect the water carried forward. Determination of water and volatile contents is also done through gravimetric methods such as the use of the Kari Fischer method. Spectroscopic methods such as nuclear magnetic resonance have been used to determine moisture in starch, cotton and other plant products. Other contamination, although rare, may be due to radioactive material during nuclear accidents (Bandaranayake, 2006; Evans, 2005;

Kibwage et al., 2005). 25

2 g Regulation of traditional medicine and products In 1991’ the world health organization (WHO) established the traditional medicine programme whose objectives were to facilitate the integration of traditional medicine into national healthcare systems, to promote the rational use of traditional medicine through the development of technical guidelines and international standards in the field of herbal medicine and acupuncture and to act as clearing house for the dissemination of information on various forms of traditional medicine

(Zhang, 1998). Back then, it was realized that developing countries have a great number of traditionally used herbal medicines and folklore knowledge about them but hardly any legislative criteria to establish these traditionally used herbal medicines as part of the drug legislation

(DeJong, 1991). To date, these objectives of the WHO have not been achieved in many countries.

The potential of traditional medicine can be realized fully if traditional healthcare practitioners and the role of traditional medicine in health systems are officially recognized through the development of national policies. Regulatory and legal framework including a code of practice, training and continuing education to enhance skills and knowledge are some of needed interventions to empower THP’s. The lack of regulation means that there are just as many fake remedies and false practitioners as there are genuine treatments (Kasilo and Trapsida, 2010).

The licensing TMP’s is important because it serves as a mechanism for the traditional healers to increase their technical knowledge and will open official communication channels between the two parallel health systems and therefore provide a way of eliciting information on the types and extend of traditional healthcare. It will also encourage conventional healthcare practitioners to learn about the wider range of factors which should be considered in the diagnosis and treatment of Patients (DeJong, 1991). 26

Most of the Americas, Europe, United States of America, Canada, Asia, the Mediterranean countries, the pacific and Australia have established laws and regulations governing the trade and practice of herbal medicine. In these countries, there is no difference between herbal medicine and chemical drugs and all are treated the same. They undergo similar procedures before registration, marketing and importation (Zhang, 1998).

Only 21 countries in Africa have put in place a legal framework for the regulation traditional medicine and products (Kasilo and Trapsida , 2010). Ghana, Guinea, Mozambique, Nigeria,

South Africa, Togo, Central Africa Republic, Chad, Equatorial Guinea, Ethiopia, Gabon,

Gambia, Tanzania and Uganda are among some of the countries in Africa that have a national policy and laws regarding traditional medicine (WHO, 2005).

In Kenya, a national policy, laws and regulations on traditional medicine, complementary and alternative medicine are being developed. There is no national programme that has been issued and no national office or expert committee has been established. Herbal medicines are not regulated and there is no herbal pharmacopoeia (WHO, 2005; AU, 2007). An act of parliament is

underway to regulate and control herbal medicine practice here in Kenya. Meanwhile, the

pharmacy and poisons board which is a statutory body established through an act of parliament

seeks to control and regulate the trade in herbal medicine.

2.9 Some active principles in medicinal plants The medicinal value of plants is due to chemical substances that produce a definite physiological

action on the human body. The most important of these bioactive compounds are alkaloids,

flavonoids, tannins, and phenolic compounds (Arwa et al., 2008; Chetri et al., 2008; Edeoga et

al., 2005). 27

2,9.1 Alkaloids Alkaloids are generally white or colourless crystalline solids and exist as salts of organic acids.

They are found in 15-30% of all flowering plants and are common in families such as Fabaceae, liliaceae, Apocynaceae, Solanaceae and Papaveraceae. Most have a bitter taste. They are frequently stored in tissues other than their site of synthesis. In plants, alkaloids protect against predators, are nitrogen reserves and act as growth regulators especially germination inhibitors.

They have been used because of their antispasmodic, antibacterial, decongestant, stimulant, antihypertensive and analgesic activities. Examples are Quinine, Morphine, Cocaine, Sparteine,

Reserpine, Ergotamine, Pilocarpine, Ephedrine and Caffeine (Arwa et al., 2008; Chetri et al.,

2008; Goodwin and Mercer, 2003; Kisangau, 1999; Kokwaro, 1993; Pengelly, 2004). Table 2.2 represents the major classes of alkaloids with their examples and pharmacological actions.

* 28

Table 2.2 Major alkaloid groups with their examples and pharmacological actions

Class Example Plant Species Pharmacologic Action

Pyndine/Piperidine Nicotine Nicotiana tabacum Adrenergic and CNS

stimulant

Lobeline Lobelia inflata Expectorant, bronchodilator

Tropane Hyoscyamine Atropa belladonna Anticholinergic

Scopolamine Datura metel Anticholinergic and CNS

depressant

Quinoline Quinine Cinchona spp Antimalarial

Isoquinoline Morphine Papaver somniferum Analgesic, narcotic

Emetine Cephaelis ipecacuanha Emetic

Quinolizidine Sparteine Sarothamnus scoparius Cardiotonic, diuretic,

oxytoxic

Pyrrolizidine Symphytine Symphytum spp Hepatotoxin

Indole Reserpine Rauwolfia serpentina Antihypertensive

Yohimbine Aspidosperma quebracho Aphrodisiac

Imidazole Pilocarpine Pilocarpus jaborandi Miotic, cholinergic •*» Alkaloidal amines Colchicine Colchicium autumnale Uric acid solvent

Ephedrine Ephedra sinica Bronchodilator

Purine alkaloids Caffeine Coffea arabica CNS Stimulant

Steroidal alkaloids Veratrine Veratrum album Antihypertensive

Adapted from Pengelly, 2004 29

2.9.2 Anthraquinones

They are yellow-brown pigments, mostly occurring as O-glycosides or C-glycosides. They are found mostly in Cassia, Senna and Aloe Species. Most are used as dyes for textiles (Pengelly,

2004). Anthraquinones have many medicinal uses. They are used as laxatives and cathartics.

The leaves and pods of Senna spp are indicated for gastric emptying before x-ray and abdominal operations. Anthraquinones are also used for dressing wounds, bums and other skin lesions.

They are useful as purgatives, for treatment of diarrhea, typhoid, peptic ulcers and stomachaches because they regulate the flow of bile and promote the activity of entire digestive process

(Kisangau et al., 2007; Kokwaro, 1993; Pengelly, 2004).

2.9.3 Glycosides They are a group of compounds having a sugar moiety attached to one or more non-sugar portions by a special bond. Most glycosides remain inactive until they are hydrolyzed in the large intestines to release the aglycone which is the active ingredient. The most common glycosides are cardiac glycosides and antihelminthic glycosides. Antihelminthic glycosides are taeniacides and are found in Albizia, Hagenia, Maesa, Myrsine and Phytolaca species.

Cardiac glycosides posses a lactone ring attached to the P position at C-17. They include Digoxin and Digitoxin from Digitalis spp. They are common in Lilliaceae, Apocynaceae and

Schrophularaceae families. They affect the dynamics and rhythm of a dysfunctional heart because they increase the speed and force of systolic contractions of the heart. They have a

Positive ionotropic effect and increase myocardial contractility. Cardiac glycosides cause an

•ncrease in atrial and ventricular myocardial excitability. They reduce the rate of atrioventricular conduction and cause an increase in vagal tone and myocardial sensitivity to vagal impulses

(Chetri et al., 2008; Kdkwaro, 1993; Pengelly, 2004). 30

2.9.4 Tannins They are the largest group of polyphenols. They are widely distributed in the bark of trees, insect galls, leaves, stems and fruit. They are glycosides of gallic acid or protocatechuic acid. They are classified as hydrolysable or condensed tannins. Hydrolysable tannins are derived from simple phenolic acids especially gallic acid, which is linked to a sugar by esterification. They break down on hydrolysis to give gallic acid and glucose or ellagic acid and glucose. They are readily soluble in water and alcohol. Condensed tannins are polymers of flavan-3-ols and flavan-3, 4- diols. On hydrolysis, they form insoluble red residues. They are partially soluble in water and alcohol.

Tannins are non crystalline compounds which produce a mild acid reaction with water. They precipitate proteins and this way, they have inhibitory effect on many enzymes. Tannins also precipitate mucus, caffeine and polysaccharides. They constrict blood vessels and this astringent action enables tannins to prevent diarrhea, stop bleeding and provide a protective coat to wounds.

Tannins are used as vermifuge and are found majorly in Acacia, Kigelia, Diospyros,

Pterocarpus, Rhizopora and Spathoda species (Kisangau, 1999; Kokwaro, 1993).

Some tannins act on arachdonic acid metabolism in leucocytes with important roles in reversing inflammations and are used in wound healing. There is a strong association between tannin content and the effects popularly attributed to wound healing and anti-inflammatory activity of the plants (Araujo et al., 2008)

2.9.5 Triterpenoids They are derived from a C 30 precursor, squalene which was first isolated from shark liver. They have a configuration similar to steroids found in plants and animals. They include phytosterols

* 31 found in Withania somnifera. They help in regulation of blood cholesterol (Kisangau, 1999;

Pengelly* 2004).

2.9.6 Saponins They are composed of one or more hydrophilic glycoside moieties combined with a lipophilic triterpene derivative. They derive their name from soapwort plant (Saponaria of

Caryophyllaceae family). They are common in Sapindaceae, Aceraceae and Hippocastanaceae.

Their active portions form colloidal solutions in water. They produce lather in shaking and precipitate cholesterol. They serve as antifeedants and protect plants from microbes and fungi

(Evans, 2005; Pengelly, 2004).

Saponins are amphipathic in nature and are used as surfactants to enhance penetration of macromolecules such as proteins through cell membranes. They are used as adjuvant in vaccines

production. Saponins are also used as detergents, oil emulsifiers, foam producers in beverages

and fire extinguishers. Saponins complex with cholesterol and can therefore be used to lower

blood cholesterol. They are precursors of important therapeutic drugs such as cortisone and contraceptive oestrogens. They have pharmacological activities such as cytotoxicity, antitumor,

antimutagenic, anti-inflammatory, antiviral, anti helminthic, antibacterial and cardiac effects

(Kareru et al., 2008; Lacaille-Dubois and Wagner, 1996; Pengelly, 2004; Watt and Brayer-

bradwijk, 1962).

2.9.7 Flavonoids They occur as yellow or white pigments in plants and are found in free state or as glycosides.

Flavonoids are responsible for the shades of yellow, orange and red in flowers (Pengelly, 2004).

The chemical structure of flavonoids is based on a C15 skeleton consisting of two benzene rings

connected by a three carbon chain. The three carbon chain is closed to form a heterocyclic ring. 32 flavonoids are products of both the shikimic and acetate pathways. The most commonly occurring flavonoids are flavones and flavonols. Flavonoids are found in high quantities in tea, onions, citrus fruits and apples (Evans, 2005; Pengelly, 2004).

Flavonoids have a protective role in the heart and circulatory system. They strengthen capillaries through membrane stabilization and are used for peripheral vascular disorders. Flavonoids have antioxidant effects and have synergistic effects with ascorbic acid. Studies have shown that flavonoids have anti-inflammatory, hepatoprotective, antiviral, antiproliferative and anticarcinogenic effects. Flavonoids also have antiallergic, antiatheromatous and antihypertensive effects. Flavonoids also prevent platelet aggregation. Examples of flavonoids with medicinal value are Rutin, Quercetin, Kaempferol and Nomilin (Akaneme 2008; Arwa, et al., 2008; Harbone, 1989; Jain et al., 2006; Middleton and Kandaswami, 1993; Pengelly, 2004).

2.9.8 Oils and fats Oils and fats may be fixed oils, essential oils, sulphur oils or resins. Fixed oils do not distill at the temperature of boiling water. They consist of molecules of fatty acids which form salt or ester with one molecule of glycerin. Fixed oils include those of Balanites and Trichilia species and are used as ointment bases and emollients. Ximemia species contain cathartic oils while fixed oils from Euphorbia species such as Ricinus communis and croton species are used as purgatives.

Essential oils are non nitrogenous compounds that are volatile and usually odourless liquids.

They are responsible for the scents of flowers or other parts of plants. Most have pleasant smell.

They regulate intestinal movements, prevent colic and are used as condiments with food. Some

poorly absorbed from gut and are used as vermifuge for round worms and hookworms for

example the oil from Chenopodium species. They are also used to treat wounds because they * hinder bacterial growth. ' 33

Sulphur oils are found in the seeds of Boscia, Capparis, Cleome and Cruciferae species. They are irritating and cause reddening and vesication of skin. They are used as carminatives in small doses and as emetics in large doses. Resins are solid fats. Gum resins are found in Boswelia,

Comiphora and Calotropsis species. Resins from Albizia and Fagara species are used for urinary tract infections, while those from ipomea as purgatives. Resins are used for wound dressing because of their adhesive quality. Some resins are poisonous and are used as arrow poison. Other resins are irritating and cause vomiting and purging if taken in large doses (Evans, 2005;

Pengelly, 2004).

2.9.9 Toxalbumins They are poisonous and irritant proteins that are found in seeds of some plants. They cause violent vomiting and inflammation of the eyes and nose. They are found in Euphorbiaceae species such as Castor oil, Croton oil and Jatropha oil (Pengelly, 2004).

2.9.10 Lignans Lignans are derived from shikimic acid-cinamic acid pathways followed by a series of enzymatic reactions to form dimeric molecules (Heittiarachchi 2006). They are compounds formed essentially by union of two molecules of a phenylpropane derivative. About 300 Lignans have

been isolated (Evans, 2005). Lignans have both antioxidant and antimicrobial activities. The

simple lignan, nor-dihydro-guaiaretic acid (NDGA) from Larrea tridenlate is -a potent antioxidant (Hettiarachchi, 2006; Pengelly, 2004).

2.9.11 Lectins They are high molecular weight polypeptides. They are carbohydrate binding proteins. They

■nteract with glycoproteins bearing polysaccharides side chains on cell membranes of animals to

cause agglutination and cytotoxicity. Lectins act as mitogens and stimulate production of

0 34 immunoglobulins. They are widespread in legumes. They may be toxic and cause impairment of tissue functions (Evans 2005; Pengelly, 2004).

2.10 Literature review of the selected plants

2.10.1 Ajuga remota Benth Family: Labiatae

Ajuga remota is found growing in grasslands of Kenya and other parts of East Africa. It is an erect rhizomatous pubescent herb. It is not eaten by insects, birds or animals. Ajuga remota is a widely used plant in African folklore medicine. The leaves are used to treat toothache. The leaf decoction is used for stomachache, diarrhea, malaria, pneumonia, leishmaniasis, liver problems, and high blood pressure (Gachathi, 1993; Kokwaro , 1993; Kuria and Muriuki, 1984; Munguti,

1997).

Various principles have been isolated from genus Ajuga. Phytoecdysteroids, neo-Clerodane

Diterpenes and Diterpenes, Anthrocyanidin-glucosides, Iridoid glycosides, Withanolides,

Triterpenes, Flavonoids, Triglycerides, Ajugarin 1, 11,111 and an aglycone with a cardenolide structure; Ajugasterone C, Ajugalactone, Cyasterone. Others are p- Ecdysone, Ergosterol-5,8

Endopoxide and Essential oils (Coll and Tandron, 2005; Kubo et al., 1976,1980,1982,1983;

Kuria and Muriuki, 1984).

The isolates from Ajuga remota have been shown to act as anabolic, analgesic, antibacterial, antiestrogenic, antifungal, anti-inflammatory, antihypertensive, antileukemic, antimycobacterial, * antioxidant, antipyretic, cardiotonic, hypoglycemic, vasorelaxant, antifeedant and insect growth inhibiting (Cantrell et al., 1999; Israili and Lyousi ,2009; Kubo et al., 1976, 1981; Kuria and

Muriuki, 1984). 35

2.10.2 Harrisonia abyssinica , Oliv Family: Simaroubaceae

Harrisonia abyssinica grows widely in Kenya. It is a much branched prickly shrub, or small tree up to 6m, sometimes climbing. It is evergreen and the bark is armed. Prickles are short and hooked. Leaves are 2-3 inches long and leaflets are in about 3 pairs, obovate-ellipytical or oval, obtuse, remotely dentate-serrate or occasionally lobed. Flowers are cream or yellow in colour.

Harrisonia abyssinica is used ethnobotanically in Africa for management of fever, oedema, pneumonia, arthritis and tuberculosis. It is also used to treat plague, nausea, vomiting, snakebite, abdominal pains, hemorrhoids, dysentery, gonorrhea and skin infections (Balde et al., 1995;

Kokwaro, 1993).

Principles isolated from Harrisonia abyssinica are Harrisonin, Obacunone, Pedonin, 12(3-

Acetoxyharrisoninneorin R, Perforaquassin A, Cycloabysinone and 3- friedelanone (Balde et al.,

1995; Rajab et al., 1997; 1999). Extracts from H. abyssinica have shown antiviral, antifungal, and cytotoxic activity. They also have anticandida and antibacterial activity (Balde et al., 1995;

Kirira et al., 2006; Maregesi et al., 2008; Runyoro et al., 2006).

2.10.3 Erythrina abyssinica, Lam

Family: Papilionaceae •*> Erythrina abyssinica is medium-sized tree, usually 5-15 m in height, deciduous, thickset, with a well-branched, rounded, spreading crown. The trunk is short and the bark is yellow-buff when fresh but otherwise grey-brown to creamy brown, deeply grooved, thickly corky and often spiny.

When damaged the tree exudes a brown, gummy sap. The leaves are compound, trifoliolate and alternate. Flowers are orange-red, up to 5 cm long. The fruits are cylindrical, woody pods, 4-16 cm long and deeply constricted between the seeds. They are densely furry, light brown in colour 36 and open to set free 1-10 shiny, red seeds with a grey-black patch. Erythrina abyssinica is the most widespread species in Africa. It is found in savannahs throughout eastern and southern

Africa (Beentje, 1994; ICRAF, 1992; Noad and Bimie, 1989). pounded parts of Erythrina abyssinica are used in steam form in Kenya to treat anthrax. The bark is boiled with goat meat for treating gonorrhea. The bark of the green stem may also be pounded and then tied into a fine piece of cloth and the liquid from it squeezed into the eyes to cure inflammation of the lids. The bark may be roasted until black, powdered, and applied to bums and general body swellings. A decoction is taken orally as an antihelminthic and to relieve abdominal pains. The roots are used to treat syphilis and the leaves to cure skin diseases in cattle

(Kokwaro, 1993).

Flavonoids from Erythrina abyssinica showed antibacterial activity while the ethanolic extracts of stem bark exhibited antiviral activity. The crude extract had antiplasmodial activities and its ethyl acetate extract of the stem bark showed antiplasmodial activity against the chloroquine- sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum ((Kamat, et al.,

1981; Vlietnck et al., 1995; Yenesew et al., 2003; Yenesew et al., 2004).

2.10.4 Ocimum suave. Wild Family: Labiatae

Ocimum suave is an erect annual, with much branched glabrous or slightly pubescent stems. The leaves are long, petiolate and ovate. The plant is about 1 -2 ft long. Many of Ocimum species are * highly aromatic and several are economically important as sources of essential oils, medicinal herbs and food flavorants (Paton et al., 1999). Ocimum suave is found in tropical Asia and in

West and Eastern Africa where its geographical distribution is limited to mountainous areas

(Mekonen et al., 2003; Tan et al., 2002). • 37

Ocimum suave has various traditional medicine uses. The Leaf decoction is used for croup, bronchopneumonia, stomachache and fever in children. It is used as a nasal decongestant. The leaves are used in treatment of skin diseases, fungal infections and tumors. Stem, twigs and roots are used for stomach complains and management of menstrual problems. The whole plant is used for treatment of gonorrhea, dysentery and diarrhea. It is also used for treatment of gout, piles and as a carminative and stimulant. In some parts of East Africa, Ocimum suave is used to treat ulcers and as a cathartic. In Ethiopia, Ocimum suave is traditionally used to relieve fever and pain (Ayensu, 1978; Kokwaro, 1993; Tan et al., 2002; Watt and Brayer-bradwijk, 1962).

The extracts induced hypothermia at high doses in mice. A possible mechanism to its antipyretic activity could be through inhibition of prostaglandin synthesis (Mekonen et al., 2003). Extracts of Ocimum suave inhibited lesion formation by HCL/Ethanol at the dose of 250mg/kg in rats.

This was through increase of mucous production and physiochemical reinforcement of the gastric mucosa or effects similar to endogenous prostaglandins (Tan et al., 2002). The components of Ocimum species have antifungal, antibacterial, insecticidal and insect repellant activities (Desta ,1993; Perez - Alonzo, et al .,1995).

2.10.5 Piliostigma thonningii (Schutn.) Milne - redhead. Family: Caesalpiniaceae.

to. Piliostigma thonningii is a shrub or tree with a few scattered branches and a light crown that grows to a height of 20 ft. The bark is fissured grey. The buds are rusty hairy and the leaves are lobed (tlie shape of a camel’s foot). The flowers are white to pink and the fruit is a large smooth flattened yellowish brown pod that turns dark brown and with fine cracks on drying (Jimoh and

Oladiji, 2005; Maundu et al., 1999). Piliostigma thonningii is widely distributed in Africa and

Asia. In Kenya, it grows on western region, the central region and the wooded grasslands and 38 woodlands of the coast region. It is found in open and often disturbed sandy areas (Maundu et al, 1999).

Piliostigma thonningii has several ethnopharmacological uses. The Leaves are used for coughs, chest complains and leprosy. They are also used for small pox, snake bite, dog bite, febrifuge and rheumatism. Stem, roots and twigs are chewed or their decoction drunk for stomach pains, dysentery, respiratory ailments, hook worm, skin diseases, gonorrhea, fever and gum inflammation. The root decoction is drunk for pain in the area of spleen and for hookworms

(Ayensu , 1978; Jimoh and Oladiji, 2005). Various preparations of parts of Piliostigma thonningii have been used to arrest bleeding, to treat fevers and bacterial infections, as a laxative, antihelminthic and as an anti-inflammatory agent (Ibewuke et al., 1997; Igoli et al., 2005).

2.11 Literature review on the selected bacteria species

2.11.1 Bacillus cere us They are large cells, often in chains and form ellipsoidal spores. They are highly motile and

hemolytic on blood agar. The spores can survive normal cooking. Bacillus cereus produce food

poisoning syndrome which is characterized by nausea, vomiting, abdominal pain and diarrhea.

Other infections caused by Bacillus cereus are endophthalmitis, bacteremia and septicemia,

fulmigant sepsis with hemolysis, meningitis, empyema, pleurisy, brain abscess, urinary tract

infections and osteomyelitis. These infections are severe especially with the

immunocompromised patients, alcoholics, drug abusers and those with neoplasias. % 2.11.2 Escherichia coli Escherichia coli are one of the five species of genus Escherichia. The others are E.blattae,

Efergusonii, E. hermanii, and E. vulneris. E coli are part of normal flora of healthy individuals. 39

Escherichia coli cause bacteremia, meningitis, diarrhoeal diseases and infections of the urinary tract.

2.11.3 Pseudomonas aeruginosa Pseudomonas aeruginosa are aerobic, non spore forming, gram negative rods which are straight or slightly curved. They are 1.5 -5 pm in length and 0.5 - 1 pm in width. They are motile due to the presence of flagella. They produce a grape like or com taco like odor. Colonies are usually spreading and flat. They have serrated edges and a metallic sheen associated with autolysis of the colonies. The colonies are bright green in colour which is formed when Pyoverdin combines with Pyocyannin. Identification is by a positive oxidase test and production of blue green, red or brown diffusible pigments on Mueller Hinton agar or other non dye containing agars.

Pseudomonas aeruginosa are able to survive in aqueous environments and are a problem in hospital environment. They cause nosocomial infections of the respiratory and urinary tracts, wounds and peritonitis. Other infections caused by pseudomonas aeruginosa are folliculitis, meningitis, community acquired pneumonia and bacteremia.

2.11.4 Staphylococcus aureus Staphylococcus aureus is a member of genus staphylococcus. They are anaerobic gram positive cocci. They are large cells usually 6-8mm in diameter and are not motile. They do not form spores and are hemolytic on blood agar. The colonies are pigmented and have a cream-yellow colour. They are smooth, entire, slightly raised and translucent.

Staphylococcus aureus is a human opportunistic pathogen and is found on skin, skin glands, mouth, intestinal tract, genitor-urinary tract, upper respiratory tract, mammary glands and mucus membranes. As a nosocomial pathogen, it is a major cause of mortality and morbidity. Most of the infections due to Staphylococcus aureus are acute and pyogenic and include skin infections 40 such as furuncles, cellulitis, impetigo and post operative wound infections. Other infections are bacteremia, pneumonia, osteomyelitis, acute endocarditis, myocarditis, pericarditis, cerebritis, meningitis, scalded skin syndrome, food Poisoning, toxic shock syndrome and chorioamnitis

(Murray et al., 2003).

2.12 Antimicrobial assays

Antimicrobial activity testing can be performed by any of the following methods:

2.12.1 Broth dilution method Serial two fold dilutions of the antimicrobial agent to be tested are prepared and placed in sterile tubes with appropriate broth that will allow for growth of the test microorganisms. Control tubes which don’t contain any antimicrobial agent are also set. After 24hour incubation, the tubes are observed for turbidity which is a show of microbial growth. The microorganisms will grow in the control tubes and any other tube without sufficient concentration of antimicrobial agent to inhibit growth.

A variation of the broth dilution is the micro broth dilution method which uses volumes in pi and test samples in pg. After incubation, microbial growth is indicated by turbidity or use of a suitable dye. In broth dilution methods, the lowest concentration of the antimicrobial agent that

•*» inhibits growth of the microorganisms is regarded as the minimum inhibitory concentration

(Hettiarachhi, 2006; Moshi et al., 2009; Suffredini et al., 2006; Wagate, 2008). * 2.12.2 Disc diffusion assay This uses cellulose discs. The discs are impregnated with the antimicrobial agent and then placed equidistantly onto the inoculated plates. The plates are incubated for a stipulated time and

^timicrobial activity is determined by measuring the diameters of zones of inhibition 41

(Hettiarachchi, 2006; Kisangau et al., 2007; Moshi et al., 2009; Mothana el al., 2008; Parekh and

Chanda, 2006).

2.12-3 Well diffusion assay Small circular wells are made in the solidified inoculated media. These wells are then filled with the antimicrobial agent to be tested. The solution is allowed to diffuse into the media and then incubated. The antimicrobial activity is determined as a measure of the zone of inhibition around each well (Hettiarachchi, 2006; Kisangau el al 2007; Mbaria et al., 2006).

2.13 Ethnopharmacological documentation of Meru central district

Ethnomedical information for Meru central district is scanty. There has been no elaborative ethnobotanical research done on the area. Studies carried out earlier provide information on the general Meru region. The Meru people have preferences for certain medicinal, fruit and ornamental plants such as Fragoropsis angolensis and Prunus africana (Barr and McGrew,

2004). Githinji (1990) recorded 37 medicinal Labiateae species in Kenya. Of these, 8 were from the Meru community. Kokwaro (1993) recorded 46 plant species of ethnomedical interest among the Meru community.

Gathirwa et al (2008) demonstrated antiplasmodial activity and safety of five medicinal plants commonly used by the Meru community for the treatment of malaria, and recommended that the two plants that had good antiplasmodial and anti-malarial properties be subjected to further chemical and biochemical investigations. A study in the area identified 86 plants in 37 families * of ethnomedical and ethnoveterinary uses (Itonga, personal communication). 42

CHAPTER THREE: MATERIALS AND METHODS

3.1 The study area

The study was carried out in Meru central district of Eastern province, Kenya. The district is an important agricultural centre for production of food and cash crop for the nation of Kenya. The average annual rainfall range is 381-2032mm. The district has forest reserves like the Imenti forest (Barr and McGrew, 2004).

Meru central district is one of the 13 districts of Eastern Province. It is on the east of Mount

Kenya. It borders Laikipia district to the West. To the south, it borders Nyeri, Kirinyaga, Embu and Meru South districts, while to the east is Tharaka district. Meru North and Isiolo districts border to the North. It is on the Latitude 0°3'45'' North and 0°2'30" south and lies on Longitude

370 and 380 east. The total area covered by the district is 2982 km . The estimated population as of 2008 was 569,992 people. Figure 3.1 shows the location of Meru central district in Kenya.

The district has 160 health facilities. The doctor/patient ratio is 1:33,259. This means that most of the health services are provided by other cadres of health personnel. Most of the facilities lack qualified health personnel. The HIV/AIDS prevalence is 38%. The distribution of health facilities is not equitable and there is inaccessibility of basic health services to the populace. The road network in the district is poor and the average distance to the nearest health facility is 7 km.

Most of the people have to walk to these facilities (GoK, 2005; 2007). The low doctor/patient ratio, the high HIV/AIDS prevalence and the inaccessibility of the health facilities to the general

Population forces many people to rely on traditional medical practitioners for their health care needs. 43

Figure3.1 Map of Kenya showing the position of Meru central district

MERU CENTRAL DISTRICT

MERU NORTH

KENYA

NYERI

□ DISTRICTNAME/BOUNDARY DIVISION NAME/BOUNDARY ' LOCATION NAME/BOUNDARY P/ADJACENT DISTRICT BOUNDRAY 44

3.1.1 Selection and collection of plant materials Ethnomedical information was obtained from authentic herbal practitioners who were identified with the help of the District Cultural and Social Officer and the Local administration. Authentic herbal medical practitioners were interviewed using an open ended semi structured questionnaire according to Martin (1995). The herbal practitioners identified the plants by their local names and gave the uses of each plant and parts including the modes of administration and dosages.

Voucher specimens were collected, identified scientifically by a plant taxonomist and deposited at the Department of Land Resource Management and Agricultural Technology (LARMAT),

University of Nairobi.

The information provided by the herbal practitioners was compared with available literature on the plants. This was done through a thorough search on the plants in both print and electronic databases. Further visits to the herbalist were done to determine the plants that had greater acceptance and usage. Selection of the plant materials for this study was based on the informant consensus among the identified herbal practitioners on their traditional uses in treating microbial infections in human. The plant material for this study was collected between the months of

September and November 2009. The selected plants are listed in table 3.1 and shown on plates

3.1-3.5. Their ethnobotanical uses, the parts used and the local names are included in the table.

* 45

Table 3.1: The plants from Meru Central District selected for study on their antimicrobial activity, cytotoxicity and phytochemical properties

Family Plant Species Local Life Part used Ethnomedical uses Name form

CAESALPINIACEAE Piliostigma thomingii Mukuura Tree Stem bark, Cough, colds, chest pains, Leaves stomachache, wounds, Menorrhagia

LAB I ATE AE Ajuga remota Kirurage Herb Leaves Pneumonia, fever, toothache, dysentery, stomachache, eye infection, tongue infection

Ocimum suave Makuri Tree Leaves Blocked nostrils, abdominal pains, sore eyes, ear infections, cough, disinfectant, insecticide

PAPILIONACEAE Erythrina abyssinica Muuti Tree Root bark, Anthrax, syphilis, gonorrhea, bums, Stem bark body swellings, colic, antidote for poisoning

SIMAROUBACEAE Harrisonia abyssinica Mutagataga Shrub Whole Stomachache, abdominal pains, plant fever, nausea, vomiting, plague, swollen testicles, dysentery, gonorrhea, tuberculosis

0 46

Plate 3.1 : Photographs of aerial parts of Harrisonia abyssinica (a), Ocimum suave (b), Erythrina abyssinica (c), Ajuga remota (d) and Piliostigma thonningii (e)

f 47

3.2 Preparation of plant extracts The plants’ parts (whole plant, stem bark, root bark, and leaves) were scrutinized for any foreign

matter or moulds and cleaned with distilled water. They were chopped into small pieces and air

dried under shade at the Department of Public Health, Pharmacology and Toxicology, University

of Nairobi. The dried plant material was ground using a laboratory mill to fine powders. This was

done in a fume chamber to protect from fumes and dust during the grinding. The powdered plant

material obtained was packed in 500gram portions and placed in clean air tight polythene papers

(Gakuya, 2001; Wagate, 2008).

3.2.1 Extraction

3.2.1.1 Water extraction Five hundred grams (500 grams) of the plant powder was extracted separately by placing in

conical flasks and distilled water was added until the powder was fully submerged. Stirring and

shaking was done to ensure proper mixing. The conical flasks were corked tightly with stoppers.

Shaking was done regularly to allow for percolation for four days. On the fifth day filtration was

done using Whatman No.l filter paper and the resultant liquid was collected in sterilized beakers

which were covered tightly in aluminium foil and stored in a refrigerator at '4°C pending freeze

drying, bioassay and antimicrobial assay.

•*> 3.2.1.2 Methanol extraction Five hundred grams (500 grams) of the plant powder was extracted separately by placing the

powder Jn conical flasks and 70% methanol added until the powder was fully submerged.

Thorough stirring was done to ensure proper mixing and then the conical flask was corked with

appropriate stopper. Thereafter, shaking was done regularly to allow percolation and extraction. 48

On the fifth day, the extracts were filtered using Whatman No. 1 filter paper into another conical flask.

Methanol was removed in a rotary evaporator and collected for recycling. The resultant viscous substance was put in sterilized beakers which were covered in aluminium foil and then stored in a refrigerator at 4°C pending freeze drying, bioassay and antimicrobial assay. Freeze drying was done by use of Virtis Bench Top 3 Model freeze drier (The Virtis Company, Newyork), courtesy of Department of Veterinary Anatomy and Physiology, University of Nairobi.

3.3 Antibacterial Activity Testing

The methanolic and aqueous extracts were screened for their antibacterial activity. Broth dilution assay was used to screen the extracts for antibacterial activity. The gram positive bacterial strains used were Staphylococcus aureus (ATCC 25923) and Bacillus cereus (ATCC 25922). The gram negative bacterial strains were Escherichia coli (ATCC 11778) and Pseudomonas aeruginosa

(ATCC 27853). They were obtained from stock cultures at the Department of Public Health,

Pharmacology, and Toxicology, University of Nairobi. The positive controls were antibiotics

Ampicillin sodium powder (Flamingo Pharmaceuticals, India) for gram positive bacteria and

Gentamycin sulphate (Shangdong Kangtai, China) for gram negative bacteria (Kisangau et al.,

2007; Koshy et al., 2009; Pavithra et al., 2010). DMSO 2% was used as negative control

(Botelho et al., 2007).

3.3.1 Bacteria quantification Standard microorganisms, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureas and Bacillus cereus were inoculated on blood agar plates from stock cultures and incubated at

•U C for 24 hours. A colony from each BA plate of the standard microorganisms was emulsified 49 in 3ml physiological saline solution in sterile tubes. Four rows of 10 tubes each with 9 ml of sterile physiological saline solution were made and each row labeled with the standard microorganism to be quantified. Ten fold serial dilutions of the standard microorganisms were made. TSA plates were inoculated with 1 ml from each tube and incubated at 37°c for 24 hours.

Colonies on the TSA plates were counted and quantified. The numbers of colony forming units were 2.2><108cfu/ml (Akinyemi et al., 2005; Koshy et al„ 2007).

3.3.2 Determination of MIC and MBC Test tube method was used in the procedure for determining the MIC and MBC. Standard microorganisms Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus cereus were inoculated on BA plates and incubated at 37°C for 24 hours. A colony of each standard microorganism was emulsified in 3ml of sterile physiological saline.

Plant extracts were weighed and mixed in 4ml of sterile Mueller Hinton broth to make a master dilution of lOOmg/ml for gram positive bacteria and 250mg/ml for gram negative bacteria. Five

(5) culture tubes containing 2ml Mueller Hinton broth each were arranged in four rows for each plant extract. Two fold serial dilutions were made from the master dilution. For gram positive bacteria, the concentrations were made as lOOmg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml, 6.25mg/ml and 3.125mg/ml. For gram negative bacteria, the concentrations were made as 250mg/ml, to. 125mg/ml, 62.5mg/ml, 31.25mg/ml, 15.625mg/ml and 7.8125mg/ml. lOOql of the test bacteria was inoculated into tubes in the respective rows. The tubes were * incubated at 37°C for 24hours. The culture tubes with the lowest dilution that showed no turbidity were recorded as MIC. This is the lowest concentration that inhibited any visible growth of bacteria. For determination of MBC, lOOpl of broth from all tubes that showed 50 turbidity was removed aseptically and then plated on TSA plates using pour plate method. The plates were incubated at 37°C for 24 hours.

After incubation the lowest concentration of the plant extracts showing no bacteria growth was recorded as the MBC. The MBC is the lowest concentration at which 99.9% or more of the initial bacteria inoculums was killed. All the tests were performed in triplicate (Akinyemi et al.,

2005; Botelho et al., 2007; Dewanjee et al., 2007; Pavithra et al., 2010; Wagate, 2008)*

* 51

3.4 Cytotoxicity Study In vitro assay of Artemia salina was used to detect cell toxicity of the plant extracts (McLaughlin and Rogers 1998; McLaughlin et al., 1991; Meyer et al., 1982).

3.4.1 Preparing the marine salt solution An electric weighing machine (Sartorius-Werke Gmbh type 2472) was used to weigh thirty three grams (33g) of marine salt. This was then put in a 1 L conical flask. Distilled water was added gradually and shaking done until all the marine salt was dissolved. Thereafter, distilled water was added to 1 L mark to make the final marine salt solution (Gakuya, 2001; Wagate, 2008).

3.4.2 Hatching the brine shrimp

An improvised shallow tank made up of a shallow rectangular plastic container having two unequal chambers and several holes on the divider was used to hatch the brine shrimp. It was filled with marine salt solution (33gm of marine salt in 1 L of distilled water). A spatula

(provided with the brine shrimp eggs) was used to scoop about 50mg of the eggs which were sprinkled on the larger compartment of the container.

About 5 mg of yeast was added to the chamber with the eggs to serve as food for the nauplii. The larger compartment with the eggs was darkened by covering it. The smaller compartment was left uncovered and illuminated by a 40 watt electric bulb. After 48 hours of exposure, the nauplii were collected on the illuminated side by use of a Pasteur pipette. These nauplii were used in the brine shrimp lethality test (Pisutthanan et al., 2004).

3.4.3 Preparing the plant extracts for the test Methanol extracts of the plants were used for the study. One hundred milligrams (lOOmg) of each extract were weighed using an analytical balance (Sartorius-Werke Gmbh type 2472). This was transferred into universal bottle and ten milliliters of marine salt solution added to dissolve. 52

An electric mixer (Vortex Reamix 2789*) at 2800rpm was used to stir the mixture in order to dissolve completely. This gave a final stock solution of 10,000pg/ml from which serial dilutions were made (Gakuya, 2001; Wagate, 2008).

3.4.4 Cytotoxicity test

From the final stock solution of each plant extract, 500pl, 50pl and 5pi were transferred to sets of graduated tubes. Ten shrimps were transferred to each tube using Pasteur pipette and marine

salt solution added to the 5ml mark. This formed three sets of dilutions of 1000pg/ml, 1 OOpg/ml

and 1 Opg/ml respectively. Each dilution was set in five tubes.

DMSO in concentrations of 0.02%, 0.2% and 2% was used as the negative control. Vincristine

sulphate (BIOCRISTIN-AQ®, Biochem, India) in concentrations of lOOOpg/ml, lOOpg/ml,

lOpg/ml and lpg/ml was used as the positive control (Bastos et al., 2009; Khan et al., 2008;

Moshi et al., 2009; Ripa et al., 2009). After 24 hours, the numbers of live nauplii were counted

and percentage mortality calculated for the plant extracts and controls. Probit analysis was used

to determine the lethal concentration (LC50) at 95% confidence interval (Finney, 1971).

* 53

3.5 Preliminary Phytochemical Screening A sample of lOOg of powdered plant extract of each species was extracted in 70% methanol for

48 hours. The extract was then filtered under vacuum. The filtrate was evaporated to dryness in a rotor evaporator at 45°c and further dried to a constant weight at 45°c in a hot air oven.

Preliminary tests were done as follows:

3.5.1 Test for alkaloids Half a gram (0.5g) of each plant extract was stirred in 2 ml of 1% aqueous hydrochloric acid and heated in a boiling water bath for 10 minutes. The mixture was filtered while hot and treated with

Dragendorff s reagent. Turbidity or precipitation indicated presence of alkaloids.

3.5.2 Test for sterols and triterpenes Half a gram (0.5g) of the extract was defatted with hexane. The residue was then extracted in dichloromethane and the solution dehydrated with magnesium sulphate anhydride. The mixture was treated with 0.5ml acetic anhydride followed by 2 drops of concentrated sulphuric acid. A gradual appearance of green blue colour was indicative of sterols while colour change from pink to purple indicated triterpenes.

3.5.3 Test for saponins Half a gram (0.5g) of the plant extract was shaken in 5ml of distilled water. Frothing that persisted for at least half an hour was indicative of saponins.

3.5.4 Test for flavonoids and flavones Two hundred milligrams (200mg) of the extract was dissolved in 4 ml of 50% methanol. The * solution was warmed and metal magnesium added. Five drops of concentrated suphuric acid was added. A red colour was observed for flavonoids and orange colour for flavones. 54

3.5.5 Test for tannins Half a gram (0.5g) of the extract was dissolved in 2ml of distilled water and filtered. Two drops of ferric chloride added to the filtrate. A blue black precipitate developed when tannins were present.

3.5.6 Test for cardiac glycosides (Keller Killian test): Hundred milligrams (lOOmg) of extract was dissolved in 1ml of glacial acetic acid containing one drop of ferric chloride solution. This was then under-layered with 1 ml of concentrated sulphuric acid. The appearance of a brown ring at the interface of the two layers with the lower acidic layer turning blue green upon standing indicated the presence of cardiac glycosides.

3.5.7 Test for anthraquinones One gram (lgm) of the extract was dissolved in 70% acetone to a final concentration of

50mg/ml. The Bontrager Test was used to test for anthraquinones. Two milliliters (2 ml) of the test sample was shaken with 4 ml of hexane to defat. The upper lipophilic layer was separated and treated with 4ml of dilute ammonia. The lower layer changed to violet, then pink and indicated the presence of anthraquinones.

Sources: Chetri et al., 2008; Fawole et al., 2009; Hettiarachchi, 2006; Kareru et al., 2008,a;

Kisangau, 1999; Kisangau et al., 2007; Ngbede, et al., 2008; Orech et al., 2005; Parekh and

Chanda, 2007; Siddiqui and Ali 1997. *

* 55

CHAPTER FOUR: RESULTS AND DISCUSSION

4.1RESULTS

4.1.1 Ethnobotanical survey This study was conducted during an ongoing project in Meru Central District where ethnobotanical survey in the area identified 86 plants in 37 families as having major ethnomedical uses. Major details of the ethnobotanical survey are published elsewhere. My discussion with the herbalists showed that they used most of the plants in combination when treating various infections. Based on this ethnobotanical survey and my discussion with the herbalists, the leaves of Ajuga remota and Ocimum suave, the stem bark and leaves of

Piliostigma thonningii, the stem bark and root bark of Erythrina abyssinica and Harrisonia abyssinica (the whole plant) were identified for the current study on their antimicrobial activity, cytotoxicity and phytochemistry.

4.1.2 Antibacterial Testing Table 4.1 and 4.2 show the activity of the bacteria species on the plant extracts. This activity was determined by the presence or absence of growth of the bacteria on the plates with various concentrations of the plant extracts after inoculation and incubation. Table 4.3 and figure 4.1 show the MIC and MBC of the plant extracts. The MIC and MBC of the test antibiotics were less than lmg/ml. Overall the MIC and MBC of both the water and methanol plant extractsavere not significantly different.

When tfie MIC and MBC of the extracts against the bacteria were compared, there was significant difference between the bacteria species. Gram negative bacteria were more resistant to the plant extracts than gram positive bacteria. There was no difference in activity between the different plants parts used. Methanol extracts of Ajuga remota, Ocimum suave, Piliostigma 56 thonningii, Erythrina abyssinica (root bark), and Harrisonia abyssinica showed activity against

Staphylococcus aureus and Bacillus cereus. The water extracts of Erythrina abyssinica root bark were active against Staphylococcus aureus and Bacillus cereus.

However, Erythrina abyssinica stem bark did not have activity against Staphylococcus aureus and bacillus cereus at the concentrations tested. Water extracts of Ocimum suave, Ajuga remota and Erythrina abyssinica roots were active against Pseudomonas aeruginosa. About 64.3% of the extracts had activity against Pseudomonas aeruginosa. Escherichia coli was the most resistant bacteria. The most active plant extract against the bacteria species was the stem bark of

Piliostigma thonningii. Erythrina abyssinica stem bark showed no activity against the test bacteria.

* 57

Table 4.1: The antibacterial activity of the aqueous and methanolic extracts of the five plants obtained from Meru Central District against gram positive bacteria ------i------Type of lOOmg/ml 50 mg/ml 25 mg/ml 12.5mg/ml 6.25mg/ml 3.125mg/ml Plant Part extract SA BC SA BC SA BC SA BC SA BC SA BC Ocimum suave Leaves Water G G G G G G G G G G G G Ocimum suave Leaves Methanol NG NG NG NG NG NG G G G G G G Piliostigma thonningii Leaves Methanol NG NG NG G G G G G G G G G Ajuga remota Leaves Methanol NG NG G NG G G G G G G G G Ajuga remota Leaves Water G NG G NG G G G G G G G G Erythrina abyssinica Root bark Methanol NG NG NG G NG G NG G NG G G G Erythrina abyssinica Root bark Water NG NG NG NG NG NG NG G NG G NG G Piliostigma thonningii Leaves Water G G G G G G G G G G G G Piliostigma thonningii Stem bark Methanol NG NG NG NG NG NG NG NG NG NG NG NG Piliostigma thonningii Stem bark Water G G G G G G G G G G G G Erythrina abyssinica Stem bark Water G G G G G G G G G G G G Erythrina abyssinica Stem bark Methanol G G G G G G G G G G G G Harrisonia abyssinica Whole plant Methanol NG NG NG NG NG NG NG NG G G G G Harrisonia abyssinica Whole plant Water G G G G G G G G G G G G

Key: BC - Bacillus cereus; G - Growth; NG - No Growth; SA - Staphylococcus aureas 58

Table 4.2: The antibacterial activity of the aqueous and methanolic extracts of the five plants obtained from Meru Central District against gram negative bacteria x Type of 250 125 62.5 31.25 15.625 7.8125 Plant part extract mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml EC PA EC PA EC PA EC PA EC PA EC PA Ocimum suave Leaves Water G NG G G G G G G G G G G Ocimum suave Leaves Methanol NG NG NG NG NG G G G G G G G

Piliostigma thonningii Leaves Methanol NG NG NG G G G G G G G G G Ajuga remota Leaves Methanol NG NG G NG G G G G G G G G Ajuga remota Leaves Water NG NG G NG G G G G G G G G Erythrina abyssinica Root bark Methanol G NG G G G G G G G G G G Erythrina abyssinica Root bark Water NG NG G G G G G G G G G G Piliostigma thonningii Leaves Water G G G G G G G G G G G G Piliostigma thonningii Stem bark Methanol NG NG NG NG NG NG G NG G G G G Piliostigma thonningii Stem bark Water G G G G G G G G G G G G Erythrina abyssinica Stem bark Water G G G G G G G G G G G G Erythrina abyssinica Stem bark Methanol G G G G G G G G G G G G Harrisonia abyssinica Whole plant Methanol NG NG G NG G G G G G G G G Harrisonia abyssinica Whole plant Water G G G G G G G G G G G G

9 Key: EC - Escherichia coli; G - Growth; NG - No Growth; PA - Pseudomonas aeruginosa 59

Figure 4.1: MIC and MBC of the 7 methanolic plants’ extracts used Ethnomedically in Meru Central district

Plant Extract Key:

O l- O . suave Leaves Water extract on P. aeruginosa A3 - A. remota Leaves Water extract on B. cereus E7 - E. abyssinica Root bark Water extract on P. aeruginosa

0 2 - 0 . suave Leaves Methanol extract on S. aureus A 4 - A. rem ota Leaves Water extract on E .coli P4 - P. thonningii Stem bark Methanol extract on S. aureus 0 3 - 0 . suave Leaves Methanol extract on E .coli A 5 - A rem ota Leaves Water extract on P. aeruginosa P5 - P. thonningii Stem bark Methanol extract on B. cereus

0 4 - 0 . suave Leaves Methanol extract on P. aeruginosa E l - E. abyssinica Root bark Methanol extract on S. aureus P6 - P. thonningii Stem bark Methanol extract on E .coli P I - P. thonningti Leaves Methanol extract on S. aureus E2 - E. abyssinica Root bark Methanol extract on B. cereus P7- P. thonningii Stem bark Methanol extract on P. aeruginosa

P2 - P. thonningii Leaves Methanol extract on E .coli E3- E. abyssinica Root bark Methanol extract on P. H I - H. abyssinica Whole plant Methanol extract on. S. aureas aeruginosa P3 - P. thonningii Leaves Methanol extract on P. aeruginosa E4 - E. abyssinica Root bark Water extract on S. aureus H 2 - H. abyssinica Whole plant ^ethanol extract on B. cereus

A1 - A. rem ota L eaves M ethanol extract o n S. aureus E5 -E. abyssinica Root bark Water extract on B. cereus H3 - H. abyssinica Whole plant Methanol extract on E .colt A 2 - A. rem ota Leaves Methanol extract on E .coli E 6 - E. abyssinica Root bark Water extract on E .coli H4 - H. abyssinica Whole plant Methanol extract on P. aeruginosa 60

Table 4.3 MIC/MBC levels of aqueous and methanolic extracts of the selected plants obtained in Meru Central District

P lant P art E x tr a c t B a c te r ia MIC MBC (m g /m l) ("* /■ ") Ocintum suave Leaves Water P. aeruginosa 250 250

Ocimum suave Leaves Methanol B .cereus 12.5 25 S. Aureus 6.25 12.5 E. coli 31.25 62.5 P. aeruginosa 31.25 62.5

Piliostigma thonningii Leaves Methanol B .cereus 100 100 S. aureus 12.5 25 E. coli 62.5 125 P. aeruginosa 62.5 125

Ajuga remota Leaves Methanol B .cereus 25 50 S. aureus 25 50 P. aeruginosa 62.5 125 E. coli 250 250

Ajuga remota Leaves Water B. cereus 25 50 E. coli 250 250 P. aeruginosa 62.5 125

Erythrina abyssinica Root bark Methanol S. aureus 3.125 6.25 B .cereus 50 100 P. aeruginosa 125 250

Erythrina abyssinica Root bark Water S. aureus 3.125 3.125 B. cereus 12.5 25 E. coli 250 250 P. aeruginosa 125 250

Piliostigma thonningii Stem bark Methanol S. aureus 3.125 6T25 B. cereus 3.125 3.125 E. coli 31.25 62.5 P. aeruginosa 15.625 31.25 * Harissonia abyssinica Whole plant methanol S. aureus 6.25 12.5 B. cereus 6.25 12.5 E. coli 250 250 P. aeruginosa 62.5 125 61

4.1.3 Brine Shrimp Lethality Test The results are represented in the tables below. Table 4.4 represents the percentage mortality of the shrimps at the various concentrations. Table 4.5 shows the lethal concentration 50 (LC50 ) of the plant extracts at 95% confidence intervals. The results of the LC50 (pg/ml) are as follows:

Ajuga remota (Leaves) 69.2, Piliostigma thonningii (Leaves) 186.45, Erythrina abyssinica (Stem

Bark) 219.1, Erythrina abyssinica (Root Bark) 232.3, Piliostigma thonningii (Stem Bark) 264.8,

Harrisonia abyssinica (Whole Plant) 299.5 and Ocimum suave (Leaves) 312.55. The LC50 of vincristine sulphate was less than 1 pg/ml and DMSO did not kill the brine shrimps at the tested concentrations.

Figure 4.2 represents the percentage brine shrimp mortality due to serial dilutions of the extracts and positive control. All the plants in the study were toxic to the brine shrimp. Ajuga remota was most toxic to the shrimp. Ajuga remota leaves and Piliostigma thonningii leaves were toxic to the brine shrimp at low concentrations of less than 200pg/ml*

* rr * 62

Table 4.4 Percentage brine shrimp mortality caused by serial dilution of methanolic extracts of

the selected plants obtained in Meru Central District

Botanical Name Local Name Parts Used Serial Dilution of the Methanol Extract ( pg/ml) 10 100 1000

Erythrirui abyssinica MUUUTI Stem bark 10 28 80

Erythrina abyssinica MUUTI Root bark 0 34 80

Harrisonia abyssinica MUTAGATAGA Whole plant 10 30 70

Piliostigma thonningii MUKUURA Stem bark 4 36 72

Piliostigma thonningii MUKUURA Leaves 0 42 82

Ajuga remota KIRURAGE Leaves 2 68 98

Ocimum suave MAKURI Leaves 0 20 80

* 63

Table 4.5 Lethal Concentration 50 (LC50) in pg/ml, and 95% Confidence (Cl) of the selected plants’ extracts obtained in Meru Central District

BOTANICAL NAME LOCAL NAME PART TESTED LC50 95% Cl

Erythrina abyssinica MUUTI Stem Bark 219.1 61.3- 1347.7

Erythrina abyssinica MUUTI Root Bark 232.3 85.4-818.5

Harrisonia abyssinica MUTAGATAGA Whole Plant 299.5 74.1 -6008.2

Piliostigma thonningii MUKUURA Leaves 186.4 67 - 652

Piliostigma thonningii MUKUURA Stem Bark 264.8 80.7- 1737.5

Ocimum suave MAKURI Leaves 312.55 111.3-

075.3

Ajuga remota KIRURAGE Leaves 69.2 26.4-179.1 64

Figure 4.2 Comparison of the percent brine shrimp mortality due to the serial dilutions of plant extracts and that due to vincristine sulphate

vincristine

e.abyssinica root

P.thonningii stem

O . suave leaf

E.abyssinicastem

A.remota leaf

P. thonningii stem

H.abyssinica whole plant

9 65

4.1.4 Preliminary Phytochemical Analysis Tannins and saponins were found in all the plant extracts. Alkaloids, flavonoids and cardiac glycosides were each found in 85.7% of the plant extracts. Sterols were present in 71.4% of the plant extracts and triterpenes were present in 28.6% of the plant extracts. Anthraquinones and flavones were absent in all the plant extracts. The results of the phytochemical testing are represented in table 4.6

4.1.4.1 Erythrina abyssinica root bark and stem bark Alkaloids, tannins, saponins, triterpenes, flavonoids and cardiac glycosides were present in the root bark and stem bark of Erythrina abyssinica. Flavones, sterols and anthraquinones were absent in the root bark and stem bark of Erythrina abyssinica.

4.1.4.2 Piliostigma thonningii stem bark and leaves This study showed the presence of alkaloids, tannins, saponins, sterols, flavonoids and cardiac glycosides in the stem bark and leaves of Piliostigma thonningii. Flavones, triterpenes and anthraquinones were absent in the stem bark and the leaves of Piliostigma thonningii.

4.1.4.3 Ajuga remota leaves This study established that Ajuga remota leaves have alkaloids, tannins, saponins, sterols, flavonoids and cardiac glycosides. Flavones, triterpenes and anthraquinones were absent on the leaves of Ajuga remota. **■

4.1.4.4 Ocimum suave leaves In this study, tannins, saponins, sterols, and flavonoids were detected in the leaves of Ocimum suave. Alkaloids, flavones, triterpenes, cardiac glycosides and anthraquinones were absent. 6 6

4.1.4.5 Harrisonia abyssinica whole plant In this study the plant was found to contain alkaloids, tannins, saponins, sterols and cardiac

glycosides. Triterpenes, flavones, flavonoids and anthraquinones were not detected in Harissonia abyssinica

Plates 4.1 and 4.2 (a and b) show some of phytochemical the results. 67

Table 4.6 Phytochemical results of the selected plants obtained in Meru Central District

Plant A lk a lo id s T a n n in s S a p o n in s S tero ls Triterpenes F la v o n es F la v o n o id s Anthraquinones C a r d ia c g ly c o sid e s Erythrina abyssinica + + + - + - + - + root bark Piliostigma + + + + - - + - + thonningii leaves Piliostigma + + + + - - + - + thonningii stem bark Harissonia abyssinica + + + + - - - - + whole plant Erythrina abyssinica + + + - + - + - + stem bark

Ocimum suave leaves - + + + - - + - - Ajuga remota leaves + + + + - - + - + 6 8

1. Erythrina abyssinica root bark 2. Erythrina abyssinica stem bark 3. Piliostigma thonningii leaves 4. Harrisonia abyssinica whole plant.5. Piliostigma thonningii stem bark 6. Ocimum suave leaves 70

4.2 DISCUSSION

4.2.1 Ethnobotanical survey This study was able to establish that the Meru have a large traditional medicine base like many communities in Kenya. There is great potential for use of these traditional remedies in treating infections due to bacteria. Knowledge on the use of the herbal medicine is passed from generation to generation among the Meru community. This concurs with what Miaron et al

(2004) observed among the Maasai community of Kenya.

Few of the herbalists kept botanical gardens and most of the medicinal plants are sourced from the wild. The area receives sufficient amounts of rainfall and has good soils that make wide range of tree species and herbs grow. For the treatment of various infections most of the herbalists favoured combination of different plants. Leaves are still the most widely used among the plant parts. This is in agreement with previous reporting in other parts of Kenya by Wagate

(2008).

4.2.2 Antibacterial testing The MIC and MBC of the antibiotics were less than 1 mg/ml. Overall the MIC and MBC of both the water and methanol plant extracts were not significantly different. This supports the use of the plants by the herbalists since many use water as solvent when needed. There was significant difference between the bacteria species. Gram positive bacteria were more susceptible to the plant extracts than gram negative bacteria. There was no difference in activity between the different plants parts used.

All the plants’ parts tested had activity against the test bacteria except the stem bark of Erythrina ubyssinica. This may imply that there is no basis for use of the stem bark of Erythrina abyssinica ln treating infections. Wagate, (2008), was also not able to detect antibacterial activity of 71

Erythrina abyssinica. However further tests are needed to confirm whether the stem bark of

Erythrina abyssinica has any antimicrobial activity.

Of significance is that 64.3% of the extracts had activity against Pseudomonas aeruginosa which is a major cause of opportunistic infections especially in hospitals. The activity of these plants towards Pseudomonas aeruginosa should be investigated further. The stem bark of Piliostigma thonningii was the most active against all the test bacteria. Ibewuke et al (1997) showed that the methanol extracts of Piliostigma thonningii inhibited the activity of Staphylococcus aureus,

Pseudomonas aeruginosa and Bacillus subtilis. This study supports those findings. Further research is needed to study the antibacterial spectrum of this plant.

4.2.3 Brine shrimp lethality test Studies show that brine shrimp assay is an excellent method for preliminary investigations of toxicity and to screen medicinal plants. Besides it has been used to detect fungal toxins, pesticide and cytotoxicity of dental materials (Alluri, et al., 2005). The technique is easily mastered, costs less and utilizes small amount of test material (McLaughlin and Rogers, 1998).

Meyer et al (1982) and Santos et al (2003) laid down the process of interpretation of the results of bioassay such that extracts are toxic where LC50 < lOOOpg/ml and non toxic when LC50

>1000pg/ml. All the plants in the study were toxic to brine shrimp with Ajuga remota being most toxic. Previous studies have shown isolates of Ajuga remota are cytotoxic and have antileukemic action (Israili and Lyousi, 2009; Wagate, 2008).

Earlier studies have shown Harissonia abyssinica is cytotoxic (Balde et al., 1995; Kirira et al.,

2006; Maregesi et al., 2008). This study showed the LC50 Harissonia abyssinica to be

299.48pg/ml. This value qualifies to be toxic (Meyer et al., 1982; Santos et al., 2003). 72

All the plants in this study were cytotoxic and this could be due to the secondary metabolites contained in these plants such as flavonoids and saponins (Akaneme, 2008; Arwa, et al., 2008;

Harbone, 1989; Jain et al., 2006; Middleton and Kandaswami, 1993; Pengelly, 2004). 73

4.2.4 Preliminary Phytochemical analysis The medicinal value of plants lies in chemical constituents that produce definite action on the human body. In this study, tannins and saponins were found in all the plant extracts. Alkaloids, flavonoids and cardiac glycosides were each found in 85.7% of the plant extracts. Sterols were present in 71.4% of the plant extracts and triterpenes were present in 28.6% of the plant extracts.

Anthraquinones and flavones were absent in all the plant extracts.

4.2.4.1 Erythrina abyssinica root bark and stem bark Alkaloids, tannins, saponins, triterpenes, flavonoids and cardiac glycosides were present in the root bark of Erythrina abyssinica. Kareru et al (2008, a) observed that Erythrina abyssinica had saponins and that about 76% of plants so far studied contain saponins. This study supports those findings. Saponins have various activities among them antibacterial, anti-inflammatory, antiviral and as surfactants. This may explain why Erythrina abyssinica root bark is used for treatment of bacterial infections such as anthrax, gonorrhea and syphilis by the Meru community.

The anti-inflammatory activity of Erythrina abyssinica is the reason why it is used by the Meru community to manage pain and inflammation associated with bums, body swellings. Alkaloids exhibit activities such as analgesic, antispasmodic and bactericidal effects. This may be useful in relieving pain and explains why the plant is used in cases of body pains, bums and infections.

Further, the presence of alkaloids in this plant may explain its uses in relieve of colic, healing of wounds and treatment of bacterial infections among the Mem community. Tannins have astringent action that enables them to constrict blood vessels, stop bleeding and provide a protective coat on wound. Presence of tannins in Erythrina abyssinica may support the use of this plant for bums and wounds. 74

Flavonoids have many activities in the human body such as anti-inflammatory, antiviral and prevention of allergies. They also have hepatoprotective activity, are antiproliferative, have anticarcinogenic properties and are useful antioxidants. Their presence in this plant explains why it may be useful in management of body swellings, antidote for poisoning and treatment of bacterial infections like gonorrhea, syphilis and anthrax. The presence of cardiac glycosides in this plant may be indicative of its activity on the heart (Araujo et al., 2008; Arwa et al., 2008;

Chetri et al., 2008; Kareru et al., 2008,a; Kisangau, 1999; Kokwaro, 1993; Jain et al., 2006;

Pengelly, 2004). However, there is no reported use of this plant for this purpose among the Meru community.

4.2.4.2 Piliostigma thonningii stem bark and leaves Previous studies have shown that the seeds of Piliostigma thonningii have saponins, flavonoids, phenolics, anthraquinones, sterols and triterpenes, and cardiac glycosides (Jimoh and Oladji,

2005). The leaves had flavonols and exhibited antimicrobial activities (Ibewuke et al, 1997).

This study was able to show the presence of alkaloids, tannins, saponins, sterols, flavonoids and cardiac glycosides. Listed among the ethnomedical uses of Piliostigma thonningii is treatment of coughs, colds and chest pains.

The presence of flavonoids and alkaloids in this plant supports its ethnomedical uses. Flavonoids have anti allergy properties and may be useful for coughs and colds. They also have antimicrobial activities and together with alkaloids may explain why this plant is useful in management of infection due to bacteria. Since tannins have astringent action, their presence in this plant may explain its use for management of wounds and menorrhagia. However more studies are needed to establish this as a fact. 75

Sterols and triterpenes are precursors of other steroids in the body which include Vitamin D and sex hormones (Akaneme, 2008). Abnormal uterine bleeding may be due to hormonal imbalance and the presence of sterols can explain the use of this plant for management of menorrhagia by the Meru community. More studies are needed to determine the type of sterols present in this plant and whether they have any role in management of hormonal disorders. The presence of cardiac glycosides may suggest that this plant has activity in the heart. It is worth noting that this is not a listed indication of this plant among the Meru community.

4.2.4.3 Ajuga remota leaves Ajuga remota has been ethnomedically used by many African communities for management of fever, toothache, malaria, dysentery, eye infections, stomachache and pneumonia (Gachathi,

1993; Kokwaro, 1993; Kuria and Muriuki, 1984). Studies done earlier have shown that this plant has glycosides, triterpenes, essential oils, flavonoids and sterols. This study established that

Ajuga remota has alkaloids, tannins, saponins, sterols, flavonoids and cardiac glycosides. Sterols are precursors of steroids in the human body and their presence in this plant mean that it may have use in treating hormonal disorders (Akaneme, 2008).

The presence of cardiac glycosides in Ajuga remota suggests that the plant may be of use in management of heart conditions. Previous study has shown that this is a possible mechanism of action (Galiwango, 1989). The presence of alkaloids, tannins, and flavonoids in this plant may explain the reason for its use in bacterial infections, relieve of pain and fever among the community (Israili and Lyousi, 2009).

4.2.4.4 Ocimum suave leaves Among the Meru community, Ocimum suave has been used for treatment of blocked nostrils, abdominal pains, sore eyes, ear infections and cough. It is also used as a disinfectant/insecticide 76

(Kokwaro, 1993). Earlier studies showed that the extracts of this plant had fever relieving properties and antiulcer activity (Desta, 1993; Perez - Alonzo, et al., 1995; Tan et al., 2002).

In this study, tannins, flavonoids, saponins, and sterols were present. The presence of saponins and flavonoids may explain the use of this plant for treatment of infections such as those of chest, eyes, stomach and nose among the Meru community. Flavonoids have antiallergic properties (Middleton and Kandaswami, 1993) and this may be the reason for use of Ocimum suave leaves in management of symptoms associated with allergies such as blocked nose and cough.

Tannins have astringent action and this is a possible explanation why this plant is used for treatment of abdominal pains (Kisangau, 1999; Kokwaro, 1993). The presence of saponins explains why this plant is used as a disinfectant/ insecticide (Kokwaro, 1993). Sterols are precursors of steroids in the body such as Vitamin D and sex hormones (Akaneme, 2008).

However, there is no recorded use of this plant among the Meru community for treatment of hormonal disorders.

4.2.4.5 Harissonia abyssinica whole plant Harissonia abyssinica is used ethnobotanically by the Meru community for management of

stomach ache, abdominal pains, fever, nausea, vomiting, plague, dysentery, gonorrhea and tuberculosis (Kokwaro, 1993). In this study Harissonia abyssinica was found to contain alkaloids, tannins, saponins, sterols and cardiac glycosides. The presence of alkaloids may be the reason why Harissonia abyssinica is used for treatment of infections due to bacteria because

some alkaloids have been shown to have antibacterial activities. Alkaloids also have antispasmodic action and this may explain the use of this plant for management of abdominal pains, nausea and vomiting. < 77

The action of alkaloids as decongestants and antibacterial lends some credence for use of this plant in management of tuberculosis (Arwa et al., 2008; Chetri et al., 2008; Pengelly, 2004).

However, more research is needed concerning the use of this plant for management of TB. The presence of alkaloids and saponins may explain why this plant is used in management of infections such as plague among the Meru community.

Since tannins are known to exhibit astringent action, the use of this plant for abdominal pains, stomachache or diarrhea is supported by the findings of this study. Sterols are precursors of steroids in the human body and their presence in this plant mean that it may have use in management of hormonal disorders (Akaneme, 2008). Principles isolated from this plant have been shown to have antibacterial, antifungal, antiviral, cytotoxic activity (Balde et al., 1995;

Kirira et al., 2006 Maregesi et al., 2008). The results of this study support these previous studies. 78

CHAPTER FIVE: GENERAL CONCLUSIONS

Due to the increase in antimicrobial resistance to the available conventional drugs, researchers are now turning to plants and other natural products as sources of drugs. The new drugs are expected to have minimal side effects, be less toxic and provide novel mechanisms of action.

Despite the fact that plants form components of every system of medicine in the world, less than

5% of over 250,000 species in the world have undergone any meaningful analysis.

Folklore medicine is a major starting point for this research as it provides a guide to the assumed properties of the plants and other natural products. However, in countries like Kenya, The subject of traditional herbal medicine is not taught in the institutions that train healthcare workers. In many instances the traditional medicine practitioners are illiterate or semi-illiterate.

It is worth noting that in countries like Belgium, Germany and France, herbal medicine is included in training of healthcare providers. In China, herbal medicine is taught in institutions like universities and there are big pharmaceutical companies that deal with herbal medicine.

For research in herbal medicine to be effective, collaboration between the herbal practitioners and conventional health care givers is of paramount importance. There is need for mutual understanding and trust between the herbal practitioners and the researchers so that this research can take root. The greatest shortcoming of herbal medicine in Kenya is that very few. natural products have undergone any scientific validation. Most of the natural products are not well understood with regard to their actions. Besides, the use of these products has not been evaluated in long term use.

Plants contain several constituents whose concentration is what determines the action of the plant. It is possible that toxicity can arise due to synergism of these constituents. Research ought 79 to be carried out to determine the accepted levels of the plants constituents that will not harm the users. This can be possible if studies of the plants are carried out in animal models to show the overall effect of the plant and that of each of its constituents. Further research can then be carried out to determine what aspects traditional herbal medicine can be incorporated in the national health care system to complement the conventional drugs.

All the plants in this study had activity against some or all the bacteria tested. The greatest activity was shown by the methanolic stem bark of Piliostigma thonningii. There were several active substances in the extracts such as alkaloids, tannins, saponins, flavonoids, sterols and cardiac glycosides. The combined action of these substances is what gives the plants their overall activity.

The fact that all the plants in this study were toxic to brine shrimp is evidence that these plants should be investigated further for their toxicity. Phytochemical screening of medicinal plants forms the basis of understanding the medicinal properties of plants. Although these plants were active against the test bacteria, their use against general bacterial infections should not be advocated for until further investigations. The herbalists should be guided by use of more scientific evaluations and studies on the plants that showed significant antibacterial activity.

•*» For now, the study was able to show some proof that the plants have antibacterial activity and that the herbalists can use them for the specific infections caused by the bacteria that were shown to be susceptible.

Recommendations

1. The plants in this study showed good antibacterial activity therefore the herbalists should

be encouraged to use them. Further pharmacologic, toxicological and phytochemical 8 0

analysis should be done on the other plants. The aim should be to identify which

constituents are responsible for the actions and at what concentrations so that they can be

isolated and made into medicine.

2. The herbalists should be encouraged to plant botanical gardens to conserve and propagate

the medicinal plants.

3. More research should be carried out to study and document the medicinal plants used by

the Meru community.

4. Policies should be put in place so that traditional medicine is taught in health training

institutions and is incorporated in the national health care system. 81

REFERENCES

Abubakar, M.G., Ukwuani, A.N and Shehu, R.A (2008). Phytochemical screenjng and

antibacterial activity of Tamarindus indica Pulp Extract. Asian Journal of Biochemistry 3(2): 134-138 Adams, M., Berset, C., Kessler, M., Hamburger, M (2009). Medicinal herhs for the treatment of rheumatic disorders - A survey of European herbals from the 1 bth 17th

century. Journal of Ethnopharmacology 121:343-359. Adenisa, S.K., Idowu, O., Ogundaini, A.O., Oludimeji, H., Olugbade, T.A. ,Onawumnj Q.O..

Paisa, M (2000). Antimicrobial constituents of the leaves of acalypha ^dk^siana and

acalypha hispida. Phytotherapy Research 14:371-374 Akaneme, F.I (2008). Identification and preliminary phytochemical analysis of herbs that can

arrest threatened miscarriage in Orba and Nsukka towns of Enugu State. African Journal

o f Biotechnology 7(1):006-011 Akinyemi, O.K., Oladapo, O., Okwara, C.E., Ibe, C.C., Fasure, K (2005). Screening Gf crude extracts of six medicinal plants used in southwest Nigerian unorthodox medicine for

methicillin resistant Staphylococcus aureus activity. B M C C om plem en tary an(j

Alternative Medicine 5:6 Alluri, V.K., Tayi, V.N.R., Dodda, S., Mulabagal, V., Hsin-Sheng, T., Gottumnkaiia y §

(2005). Assessment of bioactivity of Indian plants using brine shrimp (A r te ^ ia salin a)

lethality assay. International Journal o f Applied Science and Engineering 3 (2): j 25.134 Alonso, E ., Cerdeiras, M.P ., Femades, J ., Ferreira, F ., Moyna, P ., Soubes, M ., Va2qUeZi ^ Vero, S ., Zunino, L(1995). Screening of Uruguayan medicinal plants for antimicrobial

activity. Journal of Ethnopharmacology 45:67-70 Araujo, T.A.S., Alencar, N.L., De Amorim, E.L.C De Albuquerque, U.P (200$) \ new approach to study medicinal plants with tannins and flavonoids from the local knowledge. Journal o f Ethnopharmacology 120: 72-80 Arwa, P.S., Onyango, J.C.,and Nyunja, R.O (2008). Phytochemical compounds and

antimicrobial activity of extracts of Rhoicissus plant (Rhoicissus revoillii) (Planch) Plant Sciences Research 1 (3): 68 -73 8 2

AU (2007). The status of traditional medicine in Africa and review of the progress made on the implementation of the plan of action on the AU decade of traditional medicine 2001 - 2010. African Union, Addis Ababa Ayensu, E.S (1978). Medicinal plants of West Africa. Reference Publications, Michigan, USA Balde, A.M., Peters, L., De Bruyne, T., Geerts, S., Vanden Berghe, D., Vlietnck, A (1995).

Biological investigations of Harissonia abyssinica. Phytomedicines 14: 299-302 Bandaranayake, W.M (2006). Quality control, screening ,toxicity and regulation of herbal drugs. In: Modem Phytomedicines- Turning medicinal plants into drugs (Eds: Ahmad, I., Aqil, F., Owais,M). Willey-Vch Verlag Gmbh&Co. KGaA,Weinheim, Germany. Barquar, S.R (1995). The role of traditional medicine in rural development: in traditional medicine in Africa. Sidiga, I., Chacha, N and Kanunah, P (Eds). East African Educational Publishers Ltd Nairobi, Kenya Barr, R and McGrew, J (2004). Landscape-level tree management in Meru Central District,

Kenya: in Agroforestry in Landscapes Mosaics. Yale University Tropical Resources Institute Bastos, A.L.M., Lima, F.M.R ., Conserva, M.L ., Andrade, S.V ., Rocha, E.M.M ., Lemos, P.L.S

(2009). Studies on the antimicrobial activity and brine shrimp toxicity of Z eyheria

tuberculosa(\e 11.) bur.(bignoniaceae) extracts and their main constituents A nnals o f

Clinical Microbiology And Antimicrobials 8:16 Beentje, H. J (1994). Kenya trees, shrubs and lianas. National Museums of Kenya

Bii , C.C (2001). The potential use of Allium sativum (Garlic) for management of HIV/AIDS oral candidiasis. Abstracts of the 9th symposium of the Natural Products Research Network for Africa (NAPRECA) held at Kenyatta International Conference Centre (KICC), Nairobi, Kenya, Pg 24 Botelho, M.A., Noguera, N.A.P Bastos, G.M., Fonseca, S.G ., Lemos, T.L.G ., Montenegro, D ., Henkelbach, J ., Rao, V.S., Brito, G.A.C (2007). Antimicrobial activity of the essential

oil from Lippia sidoides, Carvacrol and Thymol against oral pathogens. B razilian Journal

o f Medical and Biological Research 40:349-356

Busia, K (2005). Medicinal provision in Africa - past and present. Phytotherapy research 19: 919-923 83

Camacho M.D.R., Philipson, S.L., Croft, P.N., Solis, S.J., Ghazanfar, S.A (2003). Screening of

plant extracts for antiprotozoal and cytotoxic activities. Journal of Ethnopharmacology 89(2-3): 185-191 Cantrell, C.L., Rajab, M.S and Franzblau, S.C (1999). Antimycobacterial Ergosterol-5, 8-

endopoxide from Ajuga remota. Planta Medica 65:732-734 Chetri, P.H., Yogol, N.S., Sherchan, J., Anupa, K.C., Mansoor, S., Thapa, P (2008) Phytochemical and antimicrobial evaluation of some medicinal plants of Nepal.

Kathmadu University Journal o f Science, Engineering and Technology 1(5): 49-54 Coll, J and Tandron, Y (2005). Isolation and identification of neo-clerodane diterpenes from

ajuga remota by high performance liquid chromatography. Phytochemical Analysis 16:61-67 Cragg, G.M and Newmann D.J (2006). Plants as source of anticancer agents, in

Ethnopharmacology,(Eds.E\aine E and Nina,N.L), in Encyclopedia of Life Support

Systems(Eolss), UNESCO, Eolss Publishers,Oxford ,Uk,(http://www.eolss.net) De Mesquita, M.L., De Paula, J.E., De Moraes, M.O., Costa-Lotufu, L.V., Grougnet, R.,

Michel, S., Tillequin, F., Espindola, L.S (2009). Cytotoxic activity of Brazilian cerrado

plants used in traditional medicine against cancer lines. Journal of Ethnopharmacology 123:439-445 DeJong, J (1991). Traditional medicine in sub-Saharan Africa: its importance and potential policy options. The World Bank. Washington Dery, B.B., Otsyina, R and Ng’atiwa (1999). Indigenous use of medicinal trees and setting priorities for their domestication in Shinyanga region, Tanzania (Nairobi, Kenya: International Centre for Research in Agro-Forestry). Desta, B (1993). Ethiopian traditional herbal drug part ii: antimicrobial activity of 63 rfffedicinal

plants. Journal of Ethnopharmacology 129-139

Dewanjee, S., Kundu, M., Maiti, A., Majumder, R., Majumder, A., Mandal, S (2007). In vitro

evaluation of antimicrobial activity of crude extract from plants Diospyros peregrine,

Coccina grandis and Swietenia macrophylla. Tropical Journal of Pharmaceutical

R esearch 6(3):773-778 Edeoga, H.O., Okwu, D.E and Mbaebie, B.O (2005). Phytochemical constituents of some

Nigerian medicinal plants. African Journal of Biotechnology 4: 685-688 84

Elujoba, A.A., Odeleye, O.M., and Ogunyemi, C.M (2005). Traditional medicine development

for medical and dental primary healthcare delivery system in Africa. African Journal of

Traditional Complementary and Alternative Medicines 2 (1):46-61 Evans, W.C (2005). Trease and Evans Pharmacognosy. 15th Edition. Elsevier Ltd, USA Farombi, E.O (2003). African indigenous plants with chemotherapeutic potentials and

biotechnological approach to the production of bioactive prophylactic agents. A frican

Journal o f Biotechnology 2 (12):662-671 Fawole, O.A., Ndhlala, A.R., Amoo, S.O., Finnie, J.F., Van-Staden, J (2009). Anti-inflammatory and phytochemical properties of twelve medicinal plants used for treating gastrointestinal

ailments in south Africa. Journal o f Ethnopharmacology 123:237-243 Finney, D.J. (1971).Probit Analysis, 3rd Ed, Cambridge University Press, Cambridge, 333 pp Gachathi, F.N (1993). Kikuyu Botanical Dictionary of Plant Names and Uses. AMREF, Nairobi, Kenya Gakuya, D.W (2001). Pharmacological and clinical evaluation of antihelminthic activity of

Abizia anthelmintica Brogn, Maerua edulis De Wolf and Maerua subcordata De Wolf

plant extracts in sheep and mice. PhD Thesis, University o f Nairobi, Nairobi, Kenya

Galiwango, B (1989). Toxicity studies of Ajuga remota benth in rats (Rattus rattus) and goats

{Capra hircus). Msc thesis, University o f Nairobi, Nairobi, Kenya Gathirwa, J.W., Rukunga, G.M., Njagi, E.N. M., Omar, S.A., Mwitari, P.G., Guantai,A.N., Tolo, F.M., Kimani, C.W., Muthaura, C.N., Kirira, P.G., Ndunda, T.N., Amalemba, G.,

Mungai, G.M., Ndiege, 1.0 (2008). The in vitro antiplasmodial and in vivo antimalarial efficacy of some medicinal plants used traditionally for treatment of malaria by the Meru

community in Kenya. Journal o f Ethnopharmacology 115:223-231 Githinji, C.W (1990).Ethnobotanical and chemotaxonomic study of some Kenyan medicinal

Labiateae species. Msc Thesis, University o f Nairobi, Nairobi, Kenya Goodwin, T.W and Mercer, E.I (2003). Introduction to Plant Biochemistry 2nd ed. Butterworth Heinemann Ltd. UK Government of Kenya (2005). Meru Central District Strategic Plan 2005-2010 Government of Kenya (2007). Meru Central District Health Plan 2007/2008 Gurib-Fakim, A (2006) Medicinal Plants: Traditions of Yesterday and Drugs for Tomorrow.

Molecular Aspects o f Medicine 27(1): 1 -93 85

Hamayun, M., Khan,S.A., Kim,H., Na, C.I., Lee, I (2006). Traditional knowledge and Ex situ conservation of some threatened medicinal plants of Swat Kohistan. Pakistan

International Journal o f Botany 2: 205-209 Hamza, Beukel, C., Matee, M.J., Moshi, M.J., Ajam, V.V., Mikx, F., Verweji, P.E (2006) Antifungal activity of some Tanzanian plants used traditionally for the treatment of fungal infections. Journal o f EthnopharmacoologyAQH (1): 124-132 Harbone, J.B (1989). Methods In Plant Biochemistry: Plant phenolics. Academic press, London, UK Hettiarachchi, D.S (2006). Isolation, Identification and Characterization of Antibacterial compounds from Carissa lanceolata R.Br. root. Msc Thesis. Curtin University, Australia Hostetmann , K., Marston ,A., Ndjoko , K ., Wolfender ,J (2000) Potential for African plants as a source of drugs. Current Organic Chemistry 4:973-1010 Ibewuke, J.C., Ogungbamila, F.O., Ogundini, A.O., Okeke, I.N., Bohlin, L (1997). Anti inflammatory and antibacterial activities of c-methyl flavonols from Piliostigma thonningii. Phytotherapy research 11:281 -284 ICRAF, (1992). A selection of useful trees and shrubs for Kenya: Notes on their identification, propagation and management for use by farming and pastoral communities. ICRAF Igoli, J.O., Ogaji, O.G., Tor-Anyin, T.A and Igoli, N.P (2005). Traditional medicine practice amongst the Igede people of Nigeria. Part ii: African Journal of Traditional Complementary and Alternative Medicine 2: 134-152 Israili, Z.H and Lyoussi, B (2009).Ethnopharmacology Of The Plants Of Genus Ajuga. Pakistan Journal of Pharmaceutical Sciences 4 (22):425-462 Iwu, M.W., Duncan, A.R and Okunji, C.O (1999). New antimicrobials of plant origin in: Janick, J (ed): Perspectives on new crops and new uses. ASHS Press, Alexandra,VA, USA.457- 462 Jain,A., Singhai, A.K and Dixit, V.K (2006). A comparative study of ethanol extract leaves of

Tephrosia purpurea pers and the flavonoid isolated for hepatoprotective activity. Indian journal of pharmaceutical science 68:740-743 Janovska, D., Kubikova, K.,and Kokoska, L. (2003). Screening for antimicrobial activity of some medicinal plant species of traditional Chinese medicine. Czech Journal of Food Science 21:107-110 86

Jimoh, F.O and Oladiji, A.T (2005). Preliminary studies on Piliostigma thonningii seeds:

Proximate analysis, mineral composition and phytochemical screening. African Journal

o f Biotechnology A (12): 1439-1442 Justin-Temu, M., Lyamuya, E.F and Makwaya, C.K (2009). Sources of antimicrobial contamination of local herbal medicines sold on the open market in Dar es Salaam,

Tanzania. East and Central African Journal o f Pharmaceutical Sciences 12(1): 19-22 Kala, C.P Farooque, N.A ., Dhar, U (2004)Prioritization of medicinal plants on the basis of available knowledge, existing practices and use value status in Utaramchal, India.

Journal o f Biodiversity and Conservation 13: 453-469 Kamat, V.S., Chuo, F.Y., Nakishi, K (1981). Antimicrobial agents from an East African plant:

Erythrina ahyssinica. Heterocycles 15: 1163-1170 Kareru, G., Gachanja,N., Keriko,M., Kenji,M (2008,b).Antimicrobial Activity of some

medicinal plants used by herbalists in Eastern Province, Kenya. African Journal of

Traditional, Complementary and Alternative Medicine 5 (1):51 -55 Kareru, G., Kenji, M., Gachanja, N., Keriko, M., Mungai, G (2007). Traditional medicine

among the Embu and Mbeere peoples of Kenya. African Journal of Traditional,

Complementary and Alternative Medicines 4:75-86 Kareru, G., Keriko, J.M., Gachanja, A.N., Kenji, G.M (2008, a). Direct detection of trterpenoid

Saponins in medicinal plants. African Journal of Traditional, complementary and

Alternative Medicines 5(1): 56-60 Kasilo, M.J and Trapsida, J (2010). Regulation of traditional medicine in the WHO African region. WHO regional office for Africa, Brazzaville, Congo Khan, A., Rahman, M., Islam, M.S (2008). Antibacterial, antifungal and cytotoxicity activity of

ambylone isolated from Amorphophallus campanulatum. Indian Jo'tirnal of

Pharmacology 40: 41-44

Kibwage, I.O., Mwangi, J.W and Thoithi, G.N (2005). Quality control of herbal medicines. East

and Central African Journal of Pharmaceutical Sciences 8(2):27- 30 Kirira, P.G., Rukunga, G.M., Wanyonyi, A.W., Muregi, F.M., Gathirwa, J.W., Muthaura, C.N., Omar, S.A., Tolo, F.M., Mungai, G.M., Ndiege, 1.0 (2006). Antiplasmodial activity and toxicity of extracts of plants used in traditional malaria therapy in Meru and Kilifi

districts of Kenya. Journal o f Ethnopharmacology 106:403-407 87

Kisangau, D. P (1999). An Ethnobotanical and phytochemical study of medicinal plants of

Makueni District. .Msc Thesis, University o f Nairobi, Nairobi, Kenya

Kisangau, D.P., Hosea, K.M., Joseph, C.C., Lyaruu, V.M (2007). Invitro antimicrobial assay of

plants used in traditional medicine in Bukoba Rural District, Tanzania. African Journal of

Traditional, Complementary and Alternative Medicines 4 (4):510-523 Kokwaro, J.O (1993). Medicinal plants of East Africa (2nd Ed.). Kenya Literature Bureau, Nairobi, Kenya Koshy, P ., Sri, N.A.M ., Wirakanain, S ., Sim, S.S ., Saravana, K ., Hong, S.L Lee, G.S ., Syarifah, N.S.A.R (2009). Antimicrobial activity of some medicinal plants from

Malaysia. American Journal o f Applied Sciences 6 (8): 1613-1617 Kubo, I., Kido, M and Fukuyama,Y (1980). X-ray crystal structure of 12-bromoajugarin-l and

conclusion on the absolute configuration of Ajugarins. Journal of the Chemical Society,

Chemical Communications 897-898 Kubo, I., Klocke, J.A., GranjianJ., Ichkawa, N and Matsumo, T (1983). Efficient isolation of phytoecydsones from Ajuga by high performance liquid chromatography and droplet

counter current chromatography. Journal o f Chromatography 257:157-161 Kubo, I., Klocke,J.A., Asano, S (1981). Insect ecdysis inhibitors from the east African medicinal

plant Ajuga remota (Labiatae). Agricultural and Biological Chemistry 45:1925-1927

Kubo, I., Klocke,J.A., Miura,I and Fukuyama,Y (1982). Structure of Ajugarin IV. Journal o f the

Chemical Society, Chemical Communications 618-619 Kubo, I., Lee,Y.W., Balogh-Nair, V., Nakanishi, K., Chapya, A (1976). Structure of Ajugarins.

Journal O f The Chemical Society, Chemical Communications 949-950

Kuria, K.A.M and Muriuki, G (1984). A new Cardiotonic agent from Ajuga remota

Benth(Labiatae).£fr.s7 Africa Medical Journal;61:533-538 - Lacaille-Dubois, M. A. and Wagner H. A., (1996). A review of the biological and

pharmacological activities of saponins. Phytomedicine, 2(4):363-386 Maregesi, S.M., Pieters, L., Ngassapa, O.D., Apers, S., Vingerhoets, R., Cos, P., Berghe, D.A., Vlietnck, A.J (2008). Screening of some Tanzanian plants from Bunda District for

antibacterial, antifungal and antiviral activities. Journal o f Ethnopharmacolgy 119: 58- 66 88

Martin, G.J (1995). Ethnobotany: A people and plants conservation manual. Chapman and Hall, London, United Kingdom Maundu, P.M ., Ngugi, G.W And Kabuye, H.S (1999). Traditional food plants of Kenya. National Museums of Kenya. Nairobi, Kenya

Mbaria, J.M., Thuva, B.S., Kaburia, H.F.A., Munenge, R and Kamau, J.M (2006). In-vitro

antimicrobial activity of Tetracera boiviniana, an ethnomedical herbal remedy used in

Malindi, Kenya. Recent Progress in Medicinal Plants 2:287-293 Mclaughlin, J.L and Rogers, L.L (1998). The use of biological assays to evaluate botanicals.

Drug Information Journal 32:513-524 McLaughlin, J.L., Chang, C.J and Smith, D.L (1991). Bench top bioassays for the discovery of

bioactive natural products: an update. In: Rhaman .{Ed),Studies in natural products

ch em istry 383-409 Mekonen, E., Bella, A., Abebe, D., Teka, F (2003). Analgesic properties of some Ethiopian

medicinal plants in different models of nociception in mice. Phytotherapy Research 17(9):1108- 1112 Meyer, B., Ferrignh ,R., Putman, E., Jacobsen ,B., Nicholes, E and Mclaughlin, L (1982). Brine

Shrimp: A convenient general bioassay for active plant constituents. Journal of Plant

R esearch 45:31-34 Miaron, O.J., Kassim, O.F and Ekaya, W.N (2004). Indigenous knowledge: the basis of Maasai

ethno veterinary diagnostic skills. Journal of Human Ecology 16(1):43- 48 Middleton, E and Kandaswaami, C (1993). The impact of plant flavonoids on mammalian biology: implications for immunity, inflammation and cancer. In Harbone, J.B(ed) The flavonoids: Advances in Research Since 1986. Chapman and Hall, London Midiwo, J.O., Yenesew, A., Juma, B.F., Omosa, K.L., Omosa, I.L., Mutisya, D~(2005). Phytochemical evaluation of some Kenyan medicinal plants. In : 11th NAPRECA symposium, Antananarivo, Madagascar Moses, N.N., James, A.M., Pierre, T., Vincent, P.K.T (2006). Study of the antibacterial activity

of the stem, bark and leaf extracts of Parkia biglobosa (jacq.) Benth. on staphylococcus

aureus. African Journal of Traditional, Complementary and Alternative Medicines 3 (2):74 -78 89

Moshi, M.J., Innocent, E., Masimba, P.J., Otieno, D.E., Weisheit, A., Mbabazi, P., Lynes, M., Meacham, K., Hamilton, A., and Urassa, I (2009). Antimicrobial and brine shrimp toxicity of some plants used in traditional medicine in Bukoba District, North Western

Tanzania. Tanzania Journal o f Health Research 2(1 ):23-29 Mothana, R.A., Salah, A.A., Sidgi, H., Faisal, M., Sama, A.Z., Ulrike, L (2008). Antimicrobial, antioxidant and cytotoxic activities and phytochemical screening of some Yemeni

medicinal plants, e CAM Advance Access: 1 -8 Munguti, K (1997) Indigenous knowledge in the management of Malaria and Visceral

Leishmaniasis among the Tugen of Kenya. Indigenous Knowledge and Development

M onitoring 5:10-12 Murray, P.R., Baron, E.J., Jorgen, J.H., Pfaller, M.A., Yolken, R.H (Eds) (2003). Manual of Clinical Microbiology. 8th Ed. ASM Press, Washinton DC, USA Musyimi, D.M., Ogur, J.A., and Muema, P.M (2008) Phytochemical compounds and

antimicrobial activity of extracts of Aspilia plant (Aspilia mosamhiecensis) (oliv) wild.

International Journal o f Botany 4(1): 56-61

Mutai, C., Bii, C., Vagias., Abates, D., Roussis, V (2009). Antimicrobial activity of A cacia

m ellifera extracts and Lupane triterpenes. Journal o f ethnopharmacology 123:143-148 Mwangi, J.W (2004). Integration of herbal medicine in national health care of developing

countries. E ast A frica M edical Journal 497-498 Mwangi, J.W., Mungai, N.N., Thoithi, G.N., Kibwage, 1.0 (2005). Traditional herbal medicine

in national health care in Kenya. East and Central African Journal of Pharmaceutical

Sciences 8(2) 22- 26 Ngbede, J ., Yakubu, R.A ., Nyam, D.A (2008). Phytochemical screening for active compounds

in Canarium schweinfurthii(atile) leaves from Jos North, Plateau State,Nigeria. R esearch

Journal of Biological Sciences 3(9): 1078-1078 Njoroge, G.N and Busman, R.W (2007). Ethnotherapeutic management of skin diseases among

the Kikuyu of Central Kenya. Journal o f Ethnopharmacology 111:303-307 Noad, T and Bimie, A (1989). Trees of Kenya. General Printers, Nairobi, Kenya Nwaougu, L.A ., Alisi, C.S ., Ibegbulem, C.O ., Igwe, C.U (2007). Phytochemical and

antimicrobial activity of ethanolic extract of Landolphia owariensis leaf. African Journal

o f Biotechnology 6(7):890-893 90

Odera, J.A (1997). "Traditional Beliefs, Sacred Groves and Home Garden Technologies” in

conservation and utilization of indigenous medicinal plants and wild relatives of food

crops, UNESCO, Nairobi, Kenya Ogwal-Okeng, J.W., Obua, C. and Anokkbonggo, W.W (2003). Acute toxicity effects of the

methanolic extract of Fagara zanthoxyloides (Lam.) root-bark. African Health Sciences 3 (3): 124-126.

Omino, E.A and Kokwaro,J.O (1991). Ethnobotany of Apocynaceae species in Kenya. Journal

of Ethnopharmacology 40:167-180 Orech, F.O., Akenga, T., Ochora,J., Friis ,H., Aagaard-Hansen, J (2005). Potential toxicity of

some leafy vegetables consumed in Nyang’oma Division, Western Kenya. A frica Journal

of Food and Nutrition Science 5 (1): 1 -13

Parekh, J and Chanda, S (2006). Invitro Antimicrobial activities of extracts of Launaea

procu m ben s Roxb. (Labiateae), Vitis vinifera L. (Vitaceae) and Cyperus rotundus L

(Cyperaceae). African Journal o f Biomedical Research 9: 89 - 93

Parekh, J and Chanda, S (2007). In Vitro antimicrobial activity and phytochemical analysis of

some Indian medicinal plants. Turkish Journal o f Biology 31:53-58 Paton, A., Harley, R.M., Harley, M.M (1999). Ocimum an overview of relationships and

classifications. In : Holm, Y., Hiltunen, R (Eds), O cim um . Medicinal and aromatic plants - industrial profiles. Harwood Academic, Amsterdam Pavithra, P.S Janani, V.S ., Charumathi, K.H ., Indumathy, R ., Sirisha, P ., Rama, S.V (2010).

Antibacterial activity of plants used in Indian herbal medicine. International Journal o f

Green Pharmacy. Downloaded on 14/6/2010 http://www.greenpharmacy.info Pengelly, A (2004) The Constituents of Medicinal Plants - An introduction to the Chemistry and Therapeutics of Herbal Medicine.2 nd Ed. CABI Publishing, USA Perez-Alonso, M.J., Velasco-Neguelera, A.. Duru, M.E., Hammander, M., Estedan, J.I (1995).

Composition of the essential oils of Ocimum basilicum and Rosamarinus officinalis from

turkey and their insecticide repellant propertiuies. Journal of Essential Oil Research 7: 73-75 Pisutthanan, S., Plianbangchang, P., Pisutthanan, N., Ruanruay, S and Muanrit, O. (2004).

Brine shrimp lethality of Thai medicinal plants in the family of Meliaceae. Naresuan

University Journal 12 (2): 13-18 91

Rajab, M.S., Fronczek, F.R., Mulholandc, D.A and Rugutt, J.K (1999). lip, 12P-

diacetoxyharissonin, a tetranortriterpenoid from Harissonia abyssinica. Phytochemistry 52:127-133 Rajab, M.S., Rugutt, J.K., Fronczek, F.R., Fischer, N.H (1997). Structural revision of harissonin

and 12P acetoxyharissonin: Two limonoids from Harrisonia abyssinica. Journal of

Natural Products 60:822-825

Ripa, F.A., Haque, M., Imran-Ul-Haque, M.D (2009). The in vitro antimicrobial, cytotoxicity

and antioxidant activity of flower extract of Saccharum spontaneum linn. E uropean

Journal o f Scientific Research 30(3):478 - 483 Runyoro, D.K.B., Matee, M.I.N., Ngassapa, O.D., Joseph,C.C., Mbwambo,Z.H (2006).

Screening of Tanzanian plants for anti- Candida activity. BMC Complementary and

Alternative Medicine. 6:11 Santos, L.P., Pinto, G.B., Takahashi, J.A., Silva, L.G., Boaventura, M.A (2003). Biological

screening of Annonaceous Bazilian Medicinal plants using Artemia salina (brine shrimp

test) - short communication. Phytomedicine 10:209-212 Sharma, V.N (2007). Essentials of pharmacology - Basic Principles and General Concepts. 3rd Ed. CBS Publishers, Newdehli, India Shukla,R., Sharma,S.B., Prabhu,K.M., Murthy,P.S (2000). Medicinal plants for treatment of

diabetes. Indian Journal o f Clinical Biochemistry 15:169-177 Siddiqui, A.A and Ali, M (1997). Practical Pharmaceutical Chemistry. 1st Ed. CBS Publishers and Distributors, New Delhi, India Sindiga, I (1995). Traditional medicine in Africa: an introduction: in Traditional medicine in Africa. Sidiga, I., Chacha, N and Kanunah, P (Eds) East African Educational Publishers Ltd Nairobi, Kenya Suffreidini, I., Mateus, P., Nepomuceno, D., Yuenes, R And Varella, A (2006). Antimicrobial

and cytotoxicity activity of Brazilian plant extracts- Clusiaceae. Memorias do Instituto

Oswaldo Cruz 101(3): 287-290 Tan, P.V., Nyasse, B., Dimo,T., Mezui, C (2002). Gastric cytoprotective anti ulcer effects of the

leaf methanol extract of Ocimum suave (Lamiacae) in rats. Journal o f

Ethnopharmacology 82:69-74 92

Van Vuuren, S.F (2008). Antimicrobial activity of South African medicinal plants. Jou rnal o f

ethnopharmacology 119:462-472 Vlietnck, A.J., Van Hoof, L., Totte, J., Lasure, A., Vanden Berghe, D., Rwagambo, P.C., Mvukiyumwani, J (1995). Screening of hundred Rwandese plants for antimicrobial and

antiviral properties. Journal of Ethnopharmacology 46: 31-37 Wagate, C.G (2008). Pharmacological evaluation of antimicrobial and bioactivity of plant used in ethnomedicine and ethnoveterinary medicine in Machakos and Kitui areas of Kenya.

Msc thesis, University o f Nairobi, Nairobi, Kenya Wanyoike, G., Chhabra, R and Omar, S (2004). Brine shrimp toxicity and antiplasmodial

activity of five Kenyan medicinal plants. Journal o f Ethnopharmacology 90(1): 129-133

Wasswa, P and Olila, D (2006). In vitro ascaricidal activity of selected indigenous plants used

in ethnoveterinary practices in Uganda. African Journal of Traditional, alternative and

Complementary Medicines 3(2):94-103 Watt, J.M and Breyer-Brandwijk, M.G (1962). The Medicinal and Poisonous plants of Southern and Eastern Africa, E and Livingstone Ltd, 2nd edition WHO (2002). Traditional Medicine: Growing needs and potential: WHO Perspectives on Medicines. World Health Organization, Geneva pp.1-6. WHO (2004). Medicinal plants- guidelines to promote patient safety and plant conservation for a US$ 60 billion industry. World Health Organization, Geneva WHO (2005). National policy on traditional medicine and regulation of herbal medicines. World Health Organization, Geneva WHO (2009). Traditional medicine. WHO website accessed on 13/7/2009 Yenesew, A., Derese, S., Irungu, B., Midiwo, O., Waters, N., Liyala, P., Hoseah, A., Heydenreich, M and Peter, G (2003). Flavonoids and Isoflavonoids with antiplasmodial

activities of the root bark of Erythrina abyssinica. Planta Medica 69:658-661 Yenesew, A., Induli, M., Derese, S., Midiwo, J.O., Heydenreich, M., Peter, G.M., Akala, H., Wangui, J., Liyala, P., Waters, C.N (2004). Antiplasmodial flavonoids from the stem

bark of Erythrina abyssinica. Phytochemistry 65:3029 - 3032 Yinegar, H and Yewhalaw, D (2007).Traditional medicinal plant knowledge and use by local

healers in Sekoru District, Jimma Zone, South Western Ethiopia. Jou rnal o f

ethnopharmacology 3:24 93

Young-Won, C., Marcy, J. B., Hee, B. C., Kinghom, A. D (2006). Drug Discovery from Natural Sources. The AAPS Journal 2006; 8 (2) Article 28 (httn://M’\vw. aupsi. ora) Zhang, X (1998). Regulatory situation of herbal medicines: a worldwide review. World Health Organization, Geneva

lUNiVE.c; 0F MlROBil 94

Appendix 1: Preparation of blood agar medium

Suspend 40g of blood agar ini liter of distilled water and boil to dissolve. Sterilize by autoclaving at 121 °c for 15 minutes. Cool the base to 45-55°c. Add 7% sterile blood to the sterile media. The blood is warmed to 37°c before adding. Dispense the mixture into sterile Petri dishes.

Appendix 2: Preparation of Mueller Hinton broth medium

Typical formula (g/l)

Beef dehydrated infusion 300

Casein hydrosylate 17.5

Starch 1.5

Dissolve 21 g in 1 liter of distilled water. Bring to boil to dissolve medium completely. Sterilize by autoclaving at 121 °c for 15minutes

Appendix 3: Preparation of Tryptone Soya Agar.

Typical formula (g/l)

Tryptone 15

Soya peptone 5

Sodium chloride 5

Agar 15

Suspend 40g in 1 liter of distilled water. Bring to boil to dissolve completely. Sterilize by autoclaving at 121°c for 15minutes